Novel biological substance nesfatin and its related substances and uses thereof

ABSTRACT

The present invention relates to a novel method of obtaining a factor involved in appetite control and/or body weight control, as well as genes obtained by said method, polypeptides encoded by said genes, or novel polypeptides obtained from the information on polypeptides encoded by said genes as a means for treating, controlling or diagnosing diseases associated with eating disorders and/or body weight control. Also the present invention relates to substances that inhibit the effects of said genes or said polypeptides as a means for treating, controlling or diagnosing diseases associated with appetite control and/or body weight control. By using thiazolidine diones having a PPAR γ agonist activity, genes and polypeptides involved in appetite regulation and/or body weight reduction can be obtained. NESFATIN or the like obtained by said method can be used as a means for treating, controlling or diagnosing diseases associated with eating disorders and/or body weight control.

FIELD OF THE INVENTION

The present invention relates to a novel method of obtaining a factorthat is involved in food intake control and/or body weight control. Thepresent invention also relates to a polypeptide obtained by said method,said polypeptide being involved in food intake control and/or bodyweight control, a nucleic acid molecule encoding said polypeptide, aswell as a means for treating, preventing and diagnosing diseasesassociated with the suppression of food intake and/or the suppression ofbody weight gain using them. The present invention also relates to ameans for treating, preventing and diagnosing diseases associated withenhanced appetite and/or enhanced body weight gain using a substancethat inhibits the effect of said polypeptide or said gene. Furthermore,the present invention relates to an animal model of diseases associatedwith food intake control and/or body weight control obtained by saidpolypeptide, said gene, and a substance that inhibits activitiesthereof, and a method of screening compounds that controls the effect orexpression of said polypeptide using the model. The present inventionalso relates to a compound that is selected by said screening method,and a diagnostic means and therapeutic agents for diseases using saidcompound.

BACKGROUND OF THE INVENTION

Obesity is a condition in which body weight, especially white adiposetissue, occurs in excess, and is generally classified as having a bodymass index (BMI) of ≧25 kg/m² and as having a body fat percentage of 25%or greater for male adults and 30% or greater for female adults. Due tohabits of eating high-fat diets and a lack of exercises in modernsociety, the percentage of people classified as obese is on the rise.According to the result of the national nutrition survey by the Ministryof Health, Labor and Welfare in 2000, the number of men who arediagnosed as obese have certainly increased during the last 10 and 20years and about 30% of men from 40-69 years old are diagnosed as obese,and for women as well, about 30% of those 60-69 years old are diagnosedas obese.

In the past, obesity was viewed as an aesthetic problem, but today,rather than obesity per se, health impairments accompanied (orpotentially accompanied) by obesity pose major clinical challenges,providing a medical grounds for the prevention and treatment of obesity.Under such circumstances, the Japan Society for the Study of Obesity(JASSO) has defined obesity as “a pathological condition that isaccompanied or suspected of being accompanied by health impairmentsresulting from or associated with obesity and that requires medicalreduction in body weight”, and proposes to deal with it as a diseaseentity. The health impairments as used herein include type 2 diabetesand impaired glucose tolerance as well as hypertension, hyperlipidemia,hyperuricemia, fatty liver, cardiac and cerebral vascular diseases,sleep apnea syndrome, orthopedic diseases such as osteoarthritis,menstrual disorders and the like (The Japanese Journal of ClinicalMedicine (Nippon Rinsho), Supplement “Adiposis”, issued by NipponRinshosha Co., Ltd. on Jul. 28, 2003). As diseases resulting fromobesity, malignant tumors are mentioned, and specifically, obesity hasbeen reported to be a risk factor for the onset of breast cancer,uterine cancer, colon cancer, kidney cancer, esophageal cancer,pancreatic cancer, liver cancer, and gallbladder cancer (The JapaneseJournal of Clinical Medicine (Nippon Rinsho), Supplement “Adiposis”,issued by Nippon Rinshosha Co., Ltd. on Jul. 28, 2003; Non-patentdocument 1, Abu-Abid et al., Journal of Medicine (USA), Jan. 1, 2002,Vol. 33, Nos. 1-4, pp. 73-86; and Nair et al., Hepatology (USA), Jul. 1,2002, Vol. 36, No. 1, pp. 150-155). In recent years, furthermore, therehas been proposed a multiple risk syndrome that increases the risk ofarteriosclerotic diseases (myocardial infarction, cerebral infarctionetc.) called “metabolic syndrome”, which is attracting attention sincecerebral vascular diseases and cardiovascular diseases account for 30%of all deaths in Japan. Therefore, the Japan Society for the Study ofObesity, the Japan Atherosclerosis Society, the Japan Diabetes Society,the Japanese Society of Hypertension, the Japanese Circulation Society,the Japanese Society of Nephrology, the Japanese Society on Thrombosisand Hemostasis, the Japanese Society of Internal Medicine collaboratedto draw up its diagnostic criteria and announced the criteria at thepress conference of the Meeting of the Japanese Society of InternalMedicine on Apr. 8, 2005. According to the criteria, with visceral fat(fat accumulation in internal organs) set at the center of the criteria,men with a waist circumference of 85 cm or greater and women with awaist circumference of 90 cm or greater who have two or more risks ofserum lipid abnormality (either one or both of a triglyceride value of150 mg/dL or more and a HDL cholesterol value of 40 mg/dL or less), highblood pressure value (either one or both of a systolic pressure of 130mmHg or more and a diastolic pressure of 85 mmHg) and high blood glucose(a fasting blood glucose level of 110 mg/dL or more) are diagnosed ashaving the metabolic syndrome (Journal of the Japanese Society ofInternal Medicine, A research committee on the diagnostic criteria formetabolic syndrome, April, 2005 issue, Vol. 94, pp. 188-203). When thiscriteria was used, it is reported, among 290 male adults who wereundergoing health screening, 61 people (21%) were diagnosed as obese,whereas 27 people (9%) were diagnosed as having the metabolic syndrome,and 9 people (3%) were not included in obesity but were diagnosed ashaving the metabolic syndrome (Igaku no Ayumi, Kazuo Takahashi andYasushi Saito, 2005, Vol. 213, No. 6, pp. 549-554).

Since the possible cause of obesity is essentially the persistent excessof energy (calorie) taken in over energy (calorie) consumed, it isrecommended that obese people or people with obesity undergo the diettherapy and/or the exercise therapy in order to lower body weight,especially body fat percentage. However, since the continuance of thesetherapies poses considerable stress on an enhanced appetite, adaptationto changes in life styles, and exercise tolerance, various difficultiesmust be overcome to continue the therapies. It is likely that when thecalorie intake was decreased in the diet therapy, the so-called reboundphenomenon, i.e., that the intestinal absorption of nutrients increasesand energy metabolism lowers, may occur, and therefore the continuanceof the diet therapy may be abandoned. Though medical treatments ofobesity include central anorectic drugs, agents promoting thermalmetabolism, absorption-inhibiting agents, steatogenesis-inhibitingagents, etc., the only agent that can be used under the health insurancesystem in Japan at present is mazindol which is classified as a centralanorexigenic drug. However, mazindol is a stimulant-like compound, andhas side effects of excitation, irritation, cardiovascular load, dysuriaetc., and the period of use has been limited to within 3 months, andthus it is not considered a drug that can be easily used (NovartisPharma KK, “Sanorex 0.5 mg tablet”, package insert).

Excessive reduction in body weight (so-called “emaciation”) or foodintake (so-called “anorexia”) concerning obesity is problematic since itcan cause infection due to a reduced defense reaction (immunity),hematopoietic disorders, amenorrhea or irregular menstruation,infertility, mental disorders, peripheral nerve paralysis, hypotension,osteoporosis etc. Generally, when BMI is <18.5 Kg/m², or men with a bodyfat percentage of 10% or less and women with a body fat percentage of15% or less are classified as emaciated. According to the result of anational nutrition survey by the Ministry of Health, Labor and Welfarein 2000, the percentage of women with BMI of <18.5 Kg/m² in the 20-39year-old bracket has risen steadily in the past 10 and 20 years, and inthe 20-29 year-old bracket about 24% are classified as “emaciated”. Thisis possibly caused by intentional reduction in food intake by youngwomen due to excessive concern over weight. However, in anorexia nervosa(food refusal), one of the central food intake disorders prevalent amongthis age group, appetite per se extremely decreases and hence thenutritional condition aggravates, sometimes leading to death due togeneral prostration. Also, as appetite-lowering diseases that includeconcepts formerly called gastroptosis, gastroatonia, or neurogenicgastritis, there is a disease termed functional dyspepsia, which is saidto exhibit symptoms of early satiety after meals and reduced appetiteetc. (Talley et al., Gut 1999, 45, Suppl. 2:1137-42). Furthermore, ascauses of anorexia, there can be mentioned cancer, inflammatorydiseases, reduced function of the pituitary, the thyroid, or the adrenaletc., post-surgery, excessive stress and the like, and persistentanorexia for a long time under these conditions may cause bodyweakening.

Under these circumstances, in recent years, vigorous research has beenunder taken on biological factors that control food intake and also onthe relationships of factors such as leptin, adiponectin and ghrelin onthe control of food intake. At present, however, no conclusions havebeen made on factors that play a leading role in food intake controland/or body weight control, and no factors such as those described abovehave yet been used in therapies. Thus, there is a strong need foridentifying factors that play a leading role in the control of foodintake and/or the control of body weight and for applying them into thetreatment of obesity and adipogenesis. However, few factors have beenreported to be involved in food intake control and/or body weightcontrol and for the PPARγ agonist widely used as a therapeutic agent fordiabetes mellitus, no direct involvement in food intake control and/orbody weight control has been reported.

On the other hand, nuclear EF-hand acidic (NEFA) is also callednucleobindin II (NUCB 2), and a polypeptide encoded by the NEFA gene hasa calcium-binding domain (EF domain) and a DNA-binding domain (Biol ChemHoppe Seyler 1994, Aug; 375(8):497-512). NEFA has a high homology withnucleobindin and is considered to be a member of the DNA-binding factorcalled the EF-hand superfamily having a reactivity with calcium(Karabinos et al., Mol Biol Evol 1996 September; 13(7):990-8). ThoughNEFA is being investigated regarding its calcium-binding ability, itsbinding with necdin, a cellular growth control factor, etc. (Kroll etal., Biochem. Biophys. Res. Commun. 1999, 24, pp. 1-8 and Tanimura etal., J. Biol. Chem. 2000, October 13:275(41):31674-81), there are noreports on its detailed functions. NEFA has been studied regarding thepossibility of being a causative gene of Usher's syndrome, anopthalmological disease, and gastric cancer (Doucet et al., Biochim.Biophys. Acta. 1998 July 1; 1407(1):84-91 and Line et al., Br. J. Cancer2002, June 5:86(11):1824-30). Furthermore, though the possibility of theNEFA polypeptide being extracellularly secreted has been demonstratedbecause it has a signal sequence at the amino terminal end (Non-patentdocument 5), there are no reports on the physiological orpharmacological role as a result of extracellular secretion thereof.Also, there are no reports that suggest a relationship between NEFA andfood intake control and/or body weight control.

OBJECTS AND SUMMARY OF THE INVENTION

Problems to be solved by the present invention are to provide a novelmethod of obtaining a factor that is involved in food intake controland/or body weight control, and to provide a gene obtained by saidmethod, a polypeptide encoded by said gene and a novel polypeptideobtained using information on the polypeptide encoded by said gene as ameans for treating, controlling and diagnosing food intake disordersand/or diseases associated with the control of body weight. It is alsoto provide a substance that inhibits the effect of said gene orpolypeptide as a means for treating, controlling and diagnosing diseasesassociated with food intake control and/or body weight control.Furthermore, it is to provide an animal model of diseases associatedwith the control of food intake and/or the control of body weightobtained by said gene or polypeptide, or a substance that inhibits them.It is also to provide a method of screening compounds that control theeffect or expression of said polypeptide using the model, compoundsselected by said screening method, and a diagnostic means andtherapeutic agents using said compounds.

After intensive and extensive study to find a novel method of obtaininga factor involved in food intake control and/or body weight control, thepresent inventors have found that, by using thiazolidine diones having aPPARγ agonist activity, genes and polypeptides involved in thesuppression of food intake and/or the reduction in body weight can beobtained. Furthermore, it was found that the factor obtained by saidmethod was NEFA whose function has not reported, and thus saidpolypeptide factor was designated as nesfatin. After investigating onnesfatin, the present inventors have found that the partial sequence forwhich no functional domains were indicated in the previous reports hasan activity on the suppression of food intake and/or the reduction inbody weight, and therefore have disclosed novel polypeptides nesfatin-1,nesfatin-1M30, nesfatin-1M16, nesfatin-1M14 and nesfatin-1M10. InNucleobindin I (NUCB1) having a high homology in the amino acid sequenceand the base sequence of the gene with NEFA/nesfatin and belonging tothe same family as the NEFA/nesfatin, it was also found, NUCB1-M30 whichis a site corresponding to the nesfatin-1M30 of NUCB1 exhibits a similaractivity.

Furthermore, it was found that an antibody that binds to nesfatin,nesfatin-1 or nesfatin-1M30 has an activity of enhancing food intake andincreasing body weight, and it was confirmed that the inhibition ofactivity of nesfatin, nesfatin-1 or nesfatin-1M30 is effective forenhancing food intake and increasing body weight.

Thus, the present invention provides the following:

(1) A method of obtaining a factor related to food intake control and/orbody weight control, said method comprising the steps of:

acting a thiazolidine dione compound having a PPARγ agonist activity toa mammalian cell, and

identifying a gene of which expression is induced by said compound;

(2) The method according to (1) wherein said thiazolidine dione compoundis troglitazone;

(3) The method according to (1) or (2) wherein said mammalian cell is anon-small cell lung cancer cell line, an adipose cell or a cerebralnerve-derived cell;

(4) The method according to (1), (2) or (3) wherein food intake controland/or body weight control is the suppression of food intake and/or thesuppression of body weight gain;

(5) A polypeptide comprising an amino acid sequence set forth in any ofSEQ ID NOs: 65-73 or SEQ ID NOs: 107-115;

(6) A polypeptide comprising an amino acid sequence set forth in any ofSEQ ID NOs: 39-41 or SEQ ID NOs: 101-103;

(7) A polypeptide comprising an amino acid sequence set forth in SEQ IDNOs: 13-15;

(8) A polypeptide comprising an amino acid sequence set forth in SEQ IDNOs: 3, 6 and 9, said polypeptide having an activity of suppressing foodintake and/or suppressing body weight gain;

(9) A polypeptide having an activity of suppressing food intake and/orsuppressing body weight gain, said polypeptide comprising an amino acidsequence having a homology of at least 60% with any of the amino acidsequence set forth in SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 and107-115; or an amino acid sequence in which some of the amino acids havebeen deleted, inserted or substituted in an amino acid sequence setforth in any of SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or 107-115;

(10) A polypeptide having an activity of suppressing food intake and/orsuppressing body weight gain, said polypeptide comprising an amino acidsequence having a homology of at least 60% with any of the amino acidsequence set forth in SEQ ID NOs: 3, 6 and 9; or an amino acid sequencein which some of the amino acids have been deleted, inserted orsubstituted in an amino acid sequence set forth in SEQ ID NO: 3, 6 or 9;or an amino acid sequence in which some of the amino acids have beendeleted, inserted or substituted in any of the amino acid sequence setforth in SEQ ID NOs: 3, 6 and 9, said polypeptide comprising at leastone recognition site for a cleaving enzyme contained in a living body inan amino acid sequence corresponding to amino acid numbers 82-162 in SEQID NO: 3, 6 or 9;

(11) The polypeptide according to any of (5) to (10) wherein at leastone amino acid has been added to the N terminal or the C terminal;

(12) The polypeptide according to any of (5) to (10) wherein at leastone amino acid residue has been modified by a compound or a peptide;

(13) The polypeptide according to any of (5) to (12) wherein saidactivity of suppressing body weight gain is an activity of suppressingbody fat gain;

(14) A nucleic acid molecule encoding a polypeptide set forth in any of(5) to (13);

(15) A nucleic acid molecule comprising a base sequence set forth in anyof SEQ ID NOs: 74-82 and 116-124;

(16) A nucleic acid molecule comprising a base sequence set forth in anyof SEQ ID NOs: 44-46 and 104-106;

(17) A nucleic acid molecule comprising a base sequence set forth in SEQID NOs: 18-20;

(18) A nucleic acid molecule that comprises a base sequence set forth inSEQ ID NO: 10, 11 or 12 and that encodes a polypeptide having anactivity of suppressing food intake and/or suppressing body weight gain;

(19) A nucleic acid molecule that hybridizes to a base sequence setforth in SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106, or 116-124, ora partial sequence thereof under a stringent condition, and that encodesa polypeptide having an activity of suppressing food intake and/orsuppressing body weight gain;

(20) The nucleic acid molecule according to any of (14) to (19) whereinsaid activity of suppressing body weight gain is an activity ofsuppressing body fat gain;

(21) A vector comprising the nucleic acid molecule according to any of(14) to (20);

(22) The vector according to (21) to which a nucleic acid molecule isoperably linked under the control of a regulatory nucleic acid moleculethat controls the expression of said nucleic acid molecule;

(23) A transformant comprising the nucleic acid molecule according toany of (14) to (20);

(24) The transformant according to (23) that expresses the transcriptionproduct of said nucleic acid molecule;

(25) The transformant according to (23) or (24) that expresses thepolypeptide encoded by said nucleic acid molecule;

(26) The transformant according to (23), (24) or (25) wherein thetransformant is a microorganism;

(27) The transformant according to (26) wherein said microorganism isEscherichia coli;

(28) The transformant according to (23), (24) or (25) wherein thetransformant is a mammalian cell;

(29) The transformant according to (23), (24) or (25) wherein thetransformant is a plant cell;

(30) A pharmaceutical composition for suppressing food intake and/orsuppressing body weight gain, said composition comprising, as an activeingredient, the polypeptide according to any of (5) to (13) or a peptidethat contains some of the amino acid sequence of said polypeptide, thevector according to (21) or (22), or the transformant according to anyof (23) to (29);

(31) The pharmaceutical composition according to (30) wherein saidactivity of suppressing body weight gain is an activity of suppressingbody fat gain;

(32) The pharmaceutical composition according to (30) or (31) forpatients with a disease selected from obesity, diabetes mellitus,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases, menstrual disorders and malignant tumors;

(33) The pharmaceutical composition according to (30) or (31) whereinthe malignant tumor is any of breast cancer, uterine cancer, coloncancer, kidney cancer, esophageal cancer, pancreatic cancer, livercancer and gallbladder cancer;

(34) The pharmaceutical composition according to any of (30) to (33)comprising a pharmaceutically acceptable additive;

(35) An antibody that binds to any polypeptide according to (5) to (13);

(36) The antibody according to (35) that binds to a peptide comprisingan amino acid sequence set forth in SEQ ID NO: 24, 32;

(37) A substance that suppresses the activity or production of thepolypeptide according to any of (5) to

(13);

(38) The substance according to (37) that suppresses the activity ofsaid polypeptide by binding to said polypeptide;

(39) The substance according to (37) wherein said substance thatsuppresses the activity of said polypeptides is the antibody accordingto (35) or (36);

(40) A substance that suppresses the expression of a gene encoding thepolypeptide according to (5) to (13);

(41) The gene expression-suppressing substance according to (40) whereinsaid gene expression-suppressing substance is an antisenseoligonucleotide molecule;

(42) The gene expression-suppressing substance according to (41) whereinthe antisense oligonucleotide molecule comprises a base sequence setforth in SEQ ID NO: 31;

(43) The gene expression-suppressing substance according to (40) whereinsaid gene expression-suppressing substance is a RNAi molecule;

(44) A vector for producing an antisense oligonucleotide molecule or aRNAi molecule, said vector comprising a nucleic acid molecule comprisinga base sequence that is complementary to the nucleic acid sequence ofthe antisense oligonucleotide molecule according to (41) or (42) or ofthe RNAi molecule according to (43);

(45) A pharmaceutical composition for enhancing appetite or enhancingbody weight gain, said composition comprising the substance according toany of (37) to (43) or the vector according to (44);

(46) The pharmaceutical composition according to (45) containing apharmaceutically acceptable additive;

(47) A transgenic non-human organism comprising the nucleic acidmolecule according to any of (14) to (20) or the vector according to anyof (21) to (22);

(48) The transgenic non-human organism according to (47) wherein thenucleic acid molecule according to any of (14) to (20) is expressed;

(49) The transgenic non-human organism according to (47) or (48) whereinthe transformant according to any of (23) to (28) has been introduced;

(50) The transgenic non-human organism according to (47), (48) or (49)wherein said transgenic non-human organism is a transgenic non-humananimal that exhibits the state of suppressed food intake or the state ofsuppressed body weight gain;

(51) The transgenic non-human organism according to (47), (48) or (49)wherein said transgenic non-human organism is a transgenic plant;

(52) A transgenic non-human animal that has introduced therein theantibody according to (35) or (36), the suppressing substance accordingto any of (37) to (39), the gene expression-suppressing substanceaccording to any of (40) to (43) or the vector according to (44), andthat exhibits the state of enhanced food intake or the state of enhancedbody weight gain;

(53) A knock-out non-human animal wherein the entire region or partthereof of a gene comprising a nucleotide sequence set forth in any ofSEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 has beendeleted;

(54) The knock-out non-human animal according to (53) that exhibitsenhanced appetite or enhanced body weight gain;

(55) The non-human animal according to (52), (53) or (54) that can beused as an animal model for a disease selected from obesity, diabetesmellitus, hypertension, hyperlipidemia, hyperuricemia, fatty liver,cardiac diseases, cerebral vascular diseases, sleep apnea syndrome,orthopedic diseases, menstrual disorders and malignant tumors;

(56) A method of producing the peptide according to any of (5) to (13)by a cell-free protein synthetic method or a chemical synthetic method;

(57) The method of producing the peptide according to any of (5) to (13)using the transformant according to any of (23) to (29), the transgenicnon-human organism according to any of (47) to (51), or the non-humananimal according to any of (52) to (55);

(58) The production method according to (56) or (57) comprising apurification process by desorption of said peptide from the antibodyaccording to (35) or (36);

(59) The production method according to (56) or (57) comprising aprocess wherein said peptide is expressed as a GST-fused protein andthen is purified using a glutathione-bound carrier;

(60) The production method according to (56) or (57) comprising aprocess wherein said peptide is expressed as a His tag-fused protein andthen is purified using a metal ion chelate carrier;

(61) The production method according to (56) or (57) comprising aprocess wherein said peptide is expressed as a FLAG tag-fused proteinand then is purified using an anti-FLAG tag antibody-bound carrier;

(62) An assay method of predicting or diagnosing the state of enhancedfood intake or enhanced body weight gain comprising the step ofdetecting the amount contained of a nucleic acid molecule comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 and 116-124, or of a polypeptide comprising an amino acidsequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15, 39-41, 65-73,101-103 and 107-115 in a biological sample from a mammal;

(63) The assay method according to (62) comprising a step of comparingthe amount contained of said nucleic acid molecule or said polypeptidein a biological sample from a mammal with that in a biological samplefrom a normal individual;

(64) The assay method according to (62) or (63) comprising a step ofjudging the state of decreased amount contained of said nucleic acidmolecule or said polypeptide in a biological sample from a mammal as astate of enhanced food intake or enhanced body weight gain;

(65) The assay method according to any of (62) to (64) comprising thestep of judging the state of decreased amount contained of said nucleicacid molecule or said polypeptide in a biological sample from a mammalas a state or a risk of developing a disease selected from obesity,diabetes mellitus, hypertension, hyperlipidemia, hyperuricemia, fattyliver, cardiac diseases, cerebral vascular diseases, sleep apneasyndrome, orthopedic diseases, menstrual disorders and malignant tumors;

(66) The assay method according to (62) or (63) comprising the step ofjudging the state of increased amount contained of said nucleic acidmolecule or said polypeptide in a biological sample from a mammal as thestate of suppressed food intake or suppressed body weight gain;

(67) The assay method according to any of (62) to (66) wherein theamount contained of said polypeptide is detected using the antibodyaccording to (35) or (36);

(68) The assay method according to any of (62) to (66) wherein theamount contained of said nucleic acid molecule is detected using atleast one of a PCR primer, a probe or a DNA chip for detecting a nucleicacid molecule comprising a nucleotide sequence set forth in any of SEQID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124;

(69) An assay kit for use in the assay method according to any of (62)to (68), said kit comprising at least one of a PCR primer, a probe or aDNA chip for detecting a nucleic acid molecule comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 and 116-124; or an antibody recognizing a polypeptide comprisingan amino acid sequence set forth in SEQ ID NOs: 3, 6, 9, 13-15, 39-41,65-73, 101-103 or 107-115, a standard peptide, or a modified peptide forthe binding competitive reaction;

(70) A method of screening a therapeutic or preventive agent having aneffect of suppressing food intake and/or suppressing body weight gain,said method comprising the steps of:

contacting a test substance with a mammalian cell, and

detecting the increased expression of a gene in said cell comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 and 116-124, or the increased amount of a polypeptideintracellularly contained in said cell or extracellularly secretedcomprising an amino acid sequence set forth in any of in SEQ ID NOs: 3,6, 9, 13-15, 39-41, 65-73, 101-103 and 107-115;

(71) The screening method according to (70) wherein the mammalian cellis one in which a regulatory nucleic acid molecule that controls theexpression of a gene comprising a nucleotide sequence set forth in anyof SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 and thenucleic acid molecule of a reporter gene have been introduced, and theinduced expression of a gene comprising a nucleotide sequence set forthin any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 isdetected by the induced expression of the reporter gene;

(72) A method of screening a therapeutic or preventive agent having aneffect of suppressing food intake and/or suppressing body weight gain,said method comprising the steps of:

administering a test substance to a mammal, and

detecting the enhanced expression of a gene comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 and 116-124 or the enhanced production of a polypeptidecomprising an amino acid sequence set forth in any of SEQ ID NOs: 3, 6,9, 13-15, 39-41, 65-73, 101-103 and 107-115 in a biological sample fromsaid test animal;

(73) A method of screening a therapeutic or preventive agent having aneffect of suppressing food intake and/or suppressing body weight gain,said method comprising the steps of:

administering a test substance to the transgenic non-human organismaccording to any of (47) to (50) or the non-human animal according toany of (52) to (55), and

detecting the suppression of food intake or the suppression of bodyweight gain in said transgenic non-human organism or said non-humananimal;

(74) The screening method according to any of (70) to (73) wherein thetherapeutic or preventive agent having an effect of suppressing foodintake and/or suppressing body weight gain is a therapeutic orpreventive agent for a disease selected from obesity, diabetes mellitus,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases, menstrual disorders and malignant tumors;

(75) The therapeutic or preventive agent having an effect of suppressingfood intake and/or suppressing body weight gain, said agent beingobtained by the method according to any of (70) to (74);

(76) The therapeutic or preventive agent according to (75) wherein thetherapeutic or preventive agent having an effect of suppressing foodintake and/or suppressing body weight gain is a therapeutic orpreventive agent for a disease selected from obesity, diabetes mellitus,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases, menstrual disorders and malignant tumors;

(77) A method of screening a therapeutic or preventive agent having aneffect of enhancing food intake and/or enhancing body weight gain, saidmethod comprising the steps of:

contacting a test substance with a mammalian cell, and

detecting the decreased expression of a gene comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 and 116-124 in said cell, or the decreased amount of apolypeptide comprising an amino acid sequence set forth in any of SEQ IDNOs: 3, 6, 9, 13-15, 39-41, 65-73, 101-103 and 107-115 intracellularlycontained in said cell or extracellularly secreted;

(78) The screening method according to (77) wherein the mammalian cellis one in which a regulatory nucleic acid molecule that controls theexpression of a gene comprising a nucleotide sequence set forth in anyof SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 and thenucleic acid molecule of a reporter gene have been introduced, and thesuppressed expression of a gene comprising a nucleotide sequence setforth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and116-124 is detected by the suppressed expression of the reporter gene;

(79) A method of screening a therapeutic or preventive agent having aneffect of enhancing food intake and/or enhancing body weight gain, saidmethod comprising the steps of:

administering a test substance to a mammal, and

detecting the suppressed expression of a gene comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 and 116-124 or the suppressed production of a polypeptidecomprising an amino acid sequence set forth in any of SEQ ID NOs: 3, 6,9, 13-15, 39-41, 65-73, 101-103 and 107-115 in a biological sample fromsaid test animal;

(80) A method of screening a therapeutic or preventive agent having aneffect of enhancing food intake and/or enhancing body weight gain, saidmethod comprising the steps of:

administering a test substance to the transgenic non-human organismaccording to any of (47) to (50) or the non-human animal according toany of (52) to (55), and

detecting the enhancement of food intake or the enhancement of bodyweight gain in said transgenic non-human organism or said non-humananimal; and

(81) The therapeutic or preventive agent having an effect of enhancingfood intake and/or enhancing body weight gain, said agent being obtainedby the method according to any of (77) to (80).

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is an image of Northern blotting using a NEFA probe, which showsthat the expression of the NEFA gene is induced by troglitazone, andthat the NEFA gene is constantly expressed in 3T3-L1 cells that weredifferentiated to precursor adipose cells, in human cerebrospinalblastocytoma cell line HBT185 cells and SQ-5 cells.

A of FIG. 2 is a drawing that shows a schematic diagram of the domainstructure of a polypeptide encoded by the NEFA gene and a figure of thesequence of the NAP peptide used for preparing anti-nesfatin antibody. Bof FIG. 2 is an image of Western blotting using a polyclonal antibodyprepared with the NAP peptide, showing that a polypeptide encoded by theNEFA gene is present in the extract of the rat brain.

FIG. 3 is an image of immunohistochemical staining using a polyclonalantibody against the NAP peptide, showing that the NEFA gene isexpressed in sites associated with food intake control such as thearcuate nucleus (Arc), paraventricular nucleus (PVN), the supraopticnucleus (SON) and the lateral hypothalamic area (LH) of the hypothalamusof the rat brain.

A of FIG. 4 is an image of Western blotting using a polyclonal antibodyagainst the NAP peptide showing the expression and the purification ofGST-NEFA which is a bound form of GST and mouse matured nesfatin. In Aof FIG. 4, lanes 1-4 represent the rainbow marker, the preinducedbacteria, the post-sonicated pellet and the purified GSTNAP,respectively. Also, B of FIG. 4 is an image of Western blotting usinganti-nesfatin antibody showing the process of GST-NEFA being cleavedwith thrombin and purified. In B of FIG. 4, lane 1 represents themarker, lane 2 represents the sample before purification, lanes 3-6represent washed samples, and lane 7 the purified sample.

FIG. 5 is a graph showing that the food intake behavior by rats issuppressed by the administration of recombinant nesfatin into the thirdventricle of the rat brain. In FIG. 5, * and ** represent a significantdifference P<0.05 and P<0.01, respectively, relative to the controlgroup.

FIG. 6 is a graph showing that the food intake behavior by rats isenhanced by the administration of anti-nesfatin antibody into the thirdventricle of the rat brain. In FIG. 6, * represents a significantdifference P<0.001 relative to the control IgG.

FIG. 7 shows an image of in situ hybridization in the brain tissue,showing that fasting can lower the expression of the nesfatin gene inthe rat hypothalamus and re-feeding can restore the expression. In FIG.7, A represents the control group, B represents the fasting group and Crepresents the re-feeding group, and the upper figures represent100-magnified images and the bottom figures represent 400-magnifiedimages.

FIG. 8 shows an image of immunohistochemical stain using anti-nesfatinantibody, showing that fasting can lower the expression of nesfatin inthe rat hypothalamus. In FIG. 8, A represents the control group and Brepresents the fasting group. It is also an image of immunohistochemicalstain (C) using anti-C-Fos antibody, showing that the reduced expressionof nesfatin during fasting is due to enhanced appetite. In FIG. 8 theupper figures represent paraventricular nucleus (PVN) and the bottomfigures represent arcuate nucleus (Arc).

FIG. 9A is a drawing that shows the amino acid sequences of human andrat nesfatin and the estimated cleavage site for prohormone convertase.V represents the estimated cleavage site for prohormone convertase.

FIG. 9B is a schematic diagram showing the position of peptides innesfatin for preparing antibody against nesfatin-1, nesfatin-2,nesfatin-3, nesfatin-2/3, as well as nesfatin-1, nesfatin-2/3 andnesfatin-3 thought to be formed by prohormone convertase.

FIG. 9C is a drawing of Western blotting showing that the antibody perse against nesfatin-1 and nesfatin-3 bind to the antigen of interest. InFIG. 9C, the drawing on the left shows the result of an experiment inwhich the nesfatin-1 peptide was migrated and subjected to Westernblotting with nesfatin-1 IgG, and the drawing on the right shows theresult of an experiment in which the nesfatin-3 peptide was migrated andsubjected to Western blotting with nesfatin C2 IgG.

FIG. 10 is an image of double immunohistochemical stain usinganti-nesfatin-1 antibody and anti-PC-1/3 antibody or anti-PC-2 antibodyshowing the presence of a cell that is simultaneously expressingnesfatin-1 and prohormone convertase (PC-1/3 or PC-2) in the rat brain.The upper panel and the lower panel of A of FIG. 10 represent thestained images with nesfatin-1 IgG in the immunohistochemical image ofthe rat hypothalamus tissue, and the upper panel in B of FIG. 10represents a fluorescent image with PC-1/3 and the lower panel in B ofFIG. 10 represents a fluorescent image with PC-2.

FIG. 11 is a graph showing that the administration of nesfatin-1 intothe third ventricle of a rat suppresses the food intake behavior of therat, but the administration of nesfatin-2 or nesfatin-3 does not causeany changes in food intake behavior.

FIG. 12 is a graph showing that the continuous administration ofnesfatin-1 into the third ventricle of a rat results in persistentsuppression in food intake behavior (A) and persistent suppression inbody weight gain (B).

FIG. 13A is a graph showing that the administration of an antibodyagainst nesfatin-1 into the ventricle of a rat enhances appetite. InFIG. 13A, * and ** represent a significant difference P<0.05 andP<0.001, respectively, relative to the control IgG-administration group.

FIG. 13B is a graph showing that the administration of a mutant in whichnesfatin-1 cannot be excised from nesfatin into the ventricle of a ratdoes not cause enhanced appetite relative to nesfatin.

FIG. 14 is a graph showing that the continuous administration ofantisense RNA against nesfatin into the ventricle of a rat results insuppression in food intake behavior (A) and suppression in body weightgain (B). In FIG. 14, * and ** represent a significant difference P<0.05and P<0.01, respectively, relative to the missense.

FIG. 15 is a graph showing the result of measuring the amount of foodintake after the administration of nesfatin-1 into the ventricle of aLean rat and a Zucker rat which is an animal model of leptin-resistantobesity. In the Zucker rat, as in the Lean rat (normal animal), thesuppression of food intake is noted by the intraventricularadministration of nesfatin-1. In FIG. 15, * and ** represent asignificant difference P<0.05 and P<0.001, respectively, relative to thephysiological saline-administration group. White boxes and hatched boxesrepresent the physiological saline-administration group and thenesfatin-1 administration group, respectively.

FIG. 16 is a graph showing the effect of intraperitoneal nesfatin-1administration into a mouse on the amount of food intake. It shows thatthe activity by nesfatin-1 of suppressing food intake can be seen in theintraperitoneal administration into the mouse as well (A). It also showsthe result when nesfatin-1 was intraperitoneally administered into anAgouti-yellow mouse which is a mouse model of obesity and the controlmouse. It shows that the intraperitoneal administration of nesfatin-1yields a similar effect in the control mouse (B) and the Agouti-yellowmouse (C) as well.

FIG. 17 is a graph showing the effect of subcutaneous nesfatin-1administration into a mouse on the amount of food intake. Thoughnesfatin-1 exhibited an effect of suppressing food intake both by theintraperitoneal administration (ip) and the subcutaneous administration(sc), the development of the effect tended to lag in the subcutaneousadministration. In FIG. 17, * and ** represent a significant differenceP<0.05 and P<0.005, respectively, relative to the physiologicalsaline-administration group.

FIG. 18A is a graph showing the effect on the amount of food intake bythe intraperitoneal administration of nesfatin-1N23, nesfatin-1M30, andnesfatin-1C29 into a mouse. Among the partial peptides of nesfatin-1,nesfatin-1M30 was only shown to exhibit the effect of suppressing foodintake. In FIG. 18A, * represents a significant difference P<0.02relative to the physiological saline-administration group.

FIG. 18B is a drawing that shows the result of amino acid alignment ofhuman, mouse and rat nesfatin-1 and the sites of nesfatin-1N23,nesfatin-1M30 and nesfatin-1C29. It was shown that amino acid sequencesare highly conserved between species in the site of nesfatin-1M30.

FIG. 19A-1 is a graph (A-1) showing a standard curve in a competitiveEIA system that determines the concentration of nesfatin or nesfatin-1in a sample, and is a table (A-2) showing the result of measurement inthe cerebrospinal fluid. The equation of the standard curve:Y=D+(A−D)/1+(Log(X)/ĈB). Plot #1 (standard value: concentration value vsmeasured value). A=8.4672E-001; B=4.3850E+000; C=3.5938E+000;D=−2.8957E-001; R̂2=0.9998.

FIG. 19A-2 is a graph (A-1) showing a standard curve in a competitiveEIA system that determines the concentration of nesfatin or nesfatin-1in a sample and is a table (A-2) showing the result of measurement inthe cerebrospinal fluid.

FIG. 19B is a graph (b-1 and b-2) showing the assay result in whichpeptide samples extracted from the hypothalamus tissue and thecerebrospinal fluid were fractionated by HPLC and nesfatin-1 in thefractions were determined by a competitive EIA system.

FIG. 20 is a graph showing the effect on the amount of food intake bythe intraperitoneal administration of partial peptides of nesfatin-1M30into a mouse. In all cases of nesfatin-1M16M (M16M), nesfatin-1M10M(M10M) or nesfatin-M14 (M14) administered, the effect of suppressingfood intake was noted. In FIG. 20, * and ** represent a significantdifference P<0.02 and P<0.002, respectively, relative to thephysiological saline-administration group.

FIG. 21A is a graph showing the effect on the amount of food intake bythe intraperitoneal administration of human nesfatin-1M30 and mouseNUCB1-M30 into a mouse. It shows that the effect of suppressing foodintake can be noted for human nesfatin-1M30 (human/nesfatin-1M30) andmouse NUCB1-M30 (mouse NUCB1) as well as mouse nesfatin-1M30(mouse/nesfatin-1M30). In FIG. 20, * represents a significant differenceP<0.02 relative to the physiological saline-administration group.

FIG. 21B is a drawing that shows the result of amino acid alignment ofhuman, rat and mouse nesfatin and human, rat and mouse NUCB1 and thesite corresponding to nesfatin-1 and the site corresponding tonesfatin-1M30. It is shown that amino acid sequences are highlyconserved at a site corresponding to nesfatin and nesfatin-1 of NUCB2,specifically nesfatin-1M30.

FIG. 21C is a drawing that shows the result of amino acid alignment ofhuman, rat and mouse nesfatin and human, rat and mouse NUCB1 and thesite corresponding to nesfatin-1 and the site corresponding tonesfatin-1M30, and represents the continuation of FIG. 21B.

FIG. 22 shows an image of in situ hybridization using a NEFA probe invarious tissues. A of FIG. 22 represents a tissue section containingparaventricular nucleus (PVN) and supraoptic nucleus (SON), B of FIG. 22represents a tissue section containing zona incerta (Zi) and arcuatenucleus (Arc), and C of FIG. 22 represents a tissue section containinglateral hypothalamic area (LHA) of the hypothalamus.

A of FIG. 23 is a graph showing that the administration of recombinantnesfatin into the third ventricle of the rat brain can suppress theamount of food intake by the rat. B of FIG. 23 is a drawing that showsthe amount of food intake during 0-1 hour, 1-3 hours, 3-6 hours and 6-12hours for the nesfatin administration group (hatched box) that received5 pmol of nesfatin into the third ventricle of the rat brain and thecontrol group (0 pmol of nesfatin, white box). In A of FIG. 23, *represents a significant difference P<0.01 relative to 0 pmol, and in Bof FIG. 23, * represents a significant difference P<0.05 and **represents a significant difference P<0.01.

A of FIG. 24 shows the result of image analysis by in situ hybridizationof the nesfatin mRNA expression at various sites of arcuate nucleus(Arc), paraventricular nucleus (PVN), lateral hypothalamic area (LHA)and supraoptic nucleus (SON) in the hypothalamus region of the rat brainin the feeding ad libitum group (control group) and those in the fastinggroup. B of FIG. 24 shows the result of image analysis by a competitiveEIA method of the expression of nesfatin-1 peptides at paraventricularnucleus (PVN) among the hypothalamic area of the rats in the feeding adlibitum group (control group: white box) and those in the fasting group(hatched box).

FIG. 25 is a drawing that shows the amount of food intake during 0-1hour, 1-3 hours, 3-6 hours and 6-12 hours for the nesfatinadministration group (hatched box) that received 5 pmol of nesfatin intothe third ventricle of the rat brain and the control group (0 pmol ofnesfatin, white box). * represents a significant difference P<0.01relative to 0 pmol.

FIG. 26 is a graph showing the amount of food intake for 1 hour afteradministration for the control group that received physiological salinealone (nesfatin-1 IgG/nesfatin-1/leptin: −/−/−), the group that receivednesfatin-1 alone (nesfatin-1 IgG/nesfatin-1/leptin: −/+/−), the groupthat received nesfatin-1 and anti-nesfatin-1 antibody (nesfatin-1IgG/nesfatin-1/leptin: +/+/−), the group that received leptin alone(nesfatin-1 IgG/nesfatin-1/leptin: −/−/+), and the group that receivedleptin and anti-nesfatin-1 antibody (nesfatin-1 IgG/nesfatin-1/leptin:+/−/+). * represents a significant difference P<0.01 relative to thecontrol group.

A of FIG. 27 shows an image of Western blotting carried out withanti-nesfatin-1 antibody using protein extracts from the rat brain. B ofFIG. 27 shows an image of Western blotting at about 47.5 kd carried outafter anti-nesfatin-1 antibody and various peptides were previouslyreacted. In the top of B of FIG. 27, the types of peptides reacted toanti-nesfatin-1 antibody were no peptides, NAP1-Ab peptide (cognatepeptide), leptin, αMSH and CART from the left, and in the bottom of B ofFIG. 27, no peptides, NAP1-Ab peptide (cognate peptide) NPY, MCH andOrexin-A from the left.

A of FIG. 28 shows an image of immunohistochemical stain using a NAPpeptide antibody in the brain tissue containing the medulla oblongata. Bof FIG. 28 shows an image of immunohistochemical stain carried out afteran antibody against the NAP peptide and the NAP peptide (cognatepeptide) were previously reacted.

A of FIG. 29 is a graph showing the body weight of the group (TGZ: +) inwhich the troglitazone-containing food was given to the normal rats(Lean) and the Zucker fa/fa rats (Zucker), and the group (TGZ: −) inwhich the troglitazone-free food was given thereto. B of FIG. 29 is agraph showing the concentration of leptin in the blood of the group(TGZ: +) in which the troglitazone-containing food was given to thenormal rats (Lean) and the Zucker fa/fa rats and the group (TGZ: −) inwhich the troglitazone-free food was given thereto. C of FIG. 29 is agraph showing the concentration of nesfatin in the brain of the group(TGZ: +) in which the troglitazone-containing food was given to thenormal rats (Lean) and the Zucker fa/fa rats and the group (TGZ: −) inwhich the troglitazone-free food was given thereto. In FIG. 29, * and **represent a significant difference P<0.05 and P<0.01, respectively,relative to the group (TGZ: −) in which the troglitazone-free food wasgiven to the normal rats (Lean), and in FIG. 29 # and ## represent asignificant difference P<0.05 and P<0.01, respectively, relative to thegroup (TGZ: −) in which the troglitazone-free food was given to theZucker fa/fa rats.

A of FIG. 30 shows an image of Western blotting of the fraction No. 45obtained by fractionating a peptide extract from the rat hypothalamus byHPLC. B of FIG. 30 shows an image of the part at a molecular weight ofabout 9.7 kd in the Western blotting image of the fraction Nos. 43-47obtained by fractionating a peptide extract from the rat hypothalamus byHPLC.

A of FIG. 31 shows the result of immunohistochemical stain using ananti-nesfatin-1 antibody in the rat brain tissue containing the arcuatenucleus, and the result of immunohistochemical stain carried out afterthe anti-nesfatin-1 antibody and various peptides were previouslyreacted. In A of FIG. 31, a-1 represents an image of immunological stainwith anti-nesfatin-1 antibody, a-2 to a-6 represent an image ofimmunological stain when the anti-nesfatin-1 antibody was previouslyreacted with the nesfatin-1 peptide (a-2), leptin (a-3), αMSH (a-4),CART (a-5) and NPY (a-6), respectively.

B of FIG. 31 shows the result of immunohistochemical stain using ananti-nesfatin-1 antibody in the rat brain tissue containing theparaventricular nucleus, and the result of immunohistochemical staincarried out after the anti-nesfatin-1 antibody and various peptides werepreviously reacted. In B, b-1 represents an image of immunological stainwith the anti-nesfatin-1 antibody, b-2 to b-6 represent an image ofimmunological stain when the anti-nesfatin-1 antibody was previouslyreacted with nesfatin-1 peptide (b-2), leptin (b-3), αMSH (b-4), CART(b-5) and NPY (b-6), respectively.

FIG. 32 is a graph of the result showing the ratio (tissue weight/bodyweight, mg/g) of tissue weight of the abdominal subcutaneous adiposetissue (A), the epididymal adipose tissue (B), the mesenteric adiposetissue (C), the retroperitoneal adipose tissue (D), the brown adiposetissue (E) and the gastrocnemial muscle (F) obtained from the rats thatwere given nesfatin-1 or physiological saline alone for 10 days relativeto the body weight of each individual. In FIG. 32, * and ** represent asignificant difference P<0.05 and P<0.005, respectively, relative to thephysiological saline-administration group.

FIG. 33 is a graph of the assay result of the amount of food intake,blood glucose, total cholesterol and triglyceride when nesfatin-1 orphysiological saline alone was intraperitoneally given to the mouse. InFIG. 33, the white box and the hatched box represent the physiologicalsaline administration group and the nesfatin-1 administration group,respectively.

In accordance with the present invention, a factor involved in foodintake control and body weight control can be obtained by using a PPARγagonist. Also, by using nesfatin, nesfatin-1, nesfatin-1M30,nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16,NUCB1-M14 and NUCB1-M10M, diseases associated with metabolic and foodintake disorders such as obesity or adiposis and nervous hyperphagia,and type 2 diabetes mellitus, impaired glucose tolerance, hypertension,hyperlipidemia, hyperuricemia, fatty liver, cardiac diseases, cerebralvascular diseases, sleep apnea syndrome, orthopedic diseases such asosteoarthritis, menstrual disorders and diseases associated withadiposis such as malignant tumors can be prevented or treated.Furthermore, by using a substance such as antibody that suppresses theactivity of nesfatin, nesfatin-1 or nesfatin-1M30, diseases associatedwith nutritional and food intake disorders such as anorexia andcibophobia in post-surgery and/or cancer patients can be prevented ortreated.

BEST MODE FOR CARRYING OUT THE INVENTION

<Method of Obtaining a Factor Involved in Food Intake Control and/orBody Weight Control>

The present invention relates to a method of obtaining a factor that isinvolved in food intake control and/or body weight control, comprisingthe steps of reacting a thiazolidine dione compound having a PPARγagonistic activity to a mammalian cell, and identifying a gene of whichexpression is induced by said compound.

As used herein “food intake control” refers to the control of the amountof food in animals or meals in humans (both are collectively referred tohereinafter as feeding etc.) during a certain period, or the control ofthe total calories taken from feeding etc. during a certain period. Thecontrol of food intake also includes the control of events such asappetite or the satiety that motivates feeding.

As used herein “the suppression of food intake” refers to the state inwhich the amount of feeding etc. or the total amount of calories takenin by feeding etc. is decreased compared to when no control of foodintake is being done, or the state in which the growing trend of theamount of feeding etc. or the total amount of calories taken in byfeeding etc. is being suppressed compared to when no control of foodintake is being done. Also, the suppression of food intake includesstates such as reduced appetite and enhanced satiety feeling.

On the other hand, “the enhancement of food intake” as used hereinrefers to the state in which the amount of feeding etc. or the totalamount of calories taken in by feeding etc. is increased compared towhen no control of food intake is being done, or the state in which thedeclining trend of the amount of feeding etc. or the total amount ofcalories taken in by feeding etc. is being suppressed compared to whenno control of food intake is being done. Also, enhanced appetiteincludes states such as an increased appetite and a suppressed feelingof satiety.

“The control of body weight” as used herein refers to controlling theabsolute body weight value, the body mass index (an index that employsbody weight and body length) or the body fat percentage. As used herein“the suppression of body weight gain” refers to the state in which theabsolute body weight value, the body mass index or the body fatpercentage are decreased or maintained compared to when no control ofbody weight is being made, or the state in which the growing trend ofthe absolute body weight value, the body mass index or the body fatpercentage is being suppressed compared to when no control of bodyweight is being made.

“The suppression of the body fat gain” as used herein refers to thestate in which the body fat percentage is decreased or maintainedcompared to when no control of body weight is being done, or the statein which the growing trend of the body fat percentage is suppressedcompared to when no control of body weight is being done. “Theenhancement of body weight gain” refers to the state in which theabsolute body weight value, the body mass index or the body fatpercentage are increased or maintained compared to when no control ofbody weight is being done, or the state in which the growing trend ofthe absolute body weight value, the body mass index or the body fatpercentage is suppressed compared to when no control of body weight isbeing done, and it is also called herein “the suppression of body weightreduction”. In the case of humans, as the representative body massindex, height BMI (body mass index) is used, which is calculated frombody weight (kg)÷height (m)÷height (m), and is expressed in units ofKg/m². Thus, the effect of such a suppression of body weight gain or anenhancement of body weight gain can also be expressed using BMI as anindex. Also, the body fat percentage is indicated by a percentage of theweight of body fat in body weight, it can be determined by the bodydensity method, the body moisture method, the body potassium-determiningmethod, the impedance method, the dual X-ray absorption method, theneutron activation method, the near infrared spectroscopic method, theskinfold thickness-determining method, the imaging method and the like(The Japanese Journal of Clinical Medicine (Nippon Rinsho) Vol. 61,Supplement 6, pp. 357-396, 2003, issued by Nippon Rinsho K.K.).

As used herein “thiazolidine dione compounds having a PPARγ agonistactivity” include, for example, troglitazone, pioglitazone,rosiglitazone, and roboglitazone etc., and troglitazone was first putinto clinical use.

Cells as used in the present invention include a non-small cell lungcancer cell line, an adipose cell and a cerebral nerve-derived cell, butthey are not limiting and any cells that express PPARγ can be used.

As the method of reacting said compound to a mammalian cell, asdescribed below, there is a method in which said cell is cultured understimulation by said compound (Satoh et al., Oncogene, England, 2002,Vol. 21, pp. 2171-2180).

The gene, of which expression is induced by the above compound, can beidentified using, for example, a method in which the gene of whichexpression was specifically induced is subjected to the subtractionmethod or the DNA array analysis and the like (Satoh et al., Oncogene,England, 2002, Vol. 21, pp. 2171-2180). In order to select a geneencoding a factor that is extracellularly secreted from among the genesthat are specifically induced by the activation of PPARγ, the nucleotidesequence of the gene is analyzed and can be selected depending onwhether the secretary signal peptide is being encoded or not.Furthermore, as the method of selecting a gene involved in food intakecontrol and/or body weight control among those genes, there can bementioned immunological detection (illustrated in Working Example 3) ora histochemical method (illustrated in Working Examples 4 and 9) with atissue extract using a brain sample containing the hypothalamus of ahuman or an animal, and an antibody that binds to a polypeptide encodedby said gene, and a method of confirming the expression in thehypothalamus using such as the in situ hybridization method (illustratedin Working Example 8) and the RT-PCR method.

For the gene thus obtained that is involved in food intake controland/or body weight control, the nucleotide sequence can be analyzed toidentify the amino acid sequence of the encoded polypeptide. A peptideor a polypeptide comprising the amino acid sequence of the polypeptideobtained or a partial amino acid sequence thereof can be prepared usinga genetic engineering method or a chemical synthetic method.

By introducing the polypeptide thus obtained or a nucleic acid moleculeencoding said peptide introduced in a form that allows in vivoexpression locally or systemically into a test animal, and then byexamining changes in the amount of food intake by said animal and/orbody weight, the polypeptide or the gene involved in food intake controland/or body weight control can be selected from among the polypeptideobtained or the gene encoding said polypeptide. In another method, byintroducing an antibody that binds to the polypeptide thus obtained oran antisense oligonucleotide molecule or a RNAi molecule capable ofsuppressing the expression of the gene encoding said polypeptide locallyor systemically into a test animal, and then by examining changes in theamount of food intake by said animal and/or body weight, the polypeptideor the gene involved in food intake control and/or body weight controlcan be selected from among the polypeptide obtained or the gene encodingsaid polypeptide.

<A Polypeptide Having an Activity of Suppressing Food Intake and/orSuppressing Body Weight Gain>

The present invention relates to a polypeptide obtained by the abovemethod, said polypeptide having an activity of suppressing food intakeand/or suppressing body weight gain. As such a polypeptide, there can bementioned a polypeptide encoded by the nesfatin gene of which thefunction had not been identified, and it was found for the first time bythe present inventors that said polypeptide has the above function. Inaccordance with the present invention, it was found that the nesfatingene is expressed in the hypothalamus of the brain that is said tocontrol appetite (illustrated in Working Examples 4, 8, 9, 24 and 26),and that the nesfatin polypeptide administered into an animal braincauses reduction in the amount of food intake and the body weight of theanimal (illustrated in Working Examples 6 and 25). It was furtherdemonstrated that, by suppressing the function of the nesfatinpolypeptide or by inhibiting the expression of the nesfatin gene, theenhancement in food intake and body weight gain can be induced inanimals (illustrated in Working Example 7 and Working Example 15).

As examples of the nesfatin polypeptide, there can be mentioned thosethat include amino acid sequences set forth in SEQ ID NOs: 3, 6 and 9. Aprecursor nesfatin polypeptide containing a human signal peptide isshown in SEQ ID NO: 2. When the precursor nesfatin polypeptide isextracellularly secreted the signal peptide is cleaved, and thus a humanmatured nesfatin polypeptide, that substantially has an activity,produces a form set forth in SEQ ID NO: 3. As used herein, the nesfatinpolypeptide is simply called nesfatin.

Furthermore, the continued intensive and extensive study on the nesfatinpolypeptide having said activity of suppressing food intake and/orsuppressing body weight gain led to the invention of a polypeptide witha novel structure having an activity of suppressing food intake and/orsuppressing body weight gain. The discovery of this polypeptide with anovel structure was based on the investigation on various peptidesderived from the nesfatin polypeptide considering a possibility that thenesfatin polypeptide may undergo cleavage by proteolytic enzymes whenthey are extracellularly secreted. As a result, it was found that apolypeptide comprising 82 amino acids having a sequence corresponding tothe amino acids No. 25 to 106 of the nesfatin polypeptide set forth inSEQ ID NO: 5 has an activity of suppressing food intake and/orsuppressing body weight gain, and reducing body fat percentage(illustrated in Working Example 12, Working Example 13 and WorkingExample 34), and that by inhibiting the suppression of function of thenesfatin-1 polypeptide, enhancement in food intake can be induced inanimals. Based on the above, said polypeptide was named nesfatin-1 (SEQID NO: 14). Though the nesfatin polypeptide has a calcium-bindingdomain, a DNA-binding domain etc. in its structure, the sequence ofnesfatin-1 polypeptide has no such existing domain structures, and thusto obtain this nesfatin-1 polypeptide could not be absolutely expectedfrom the conventional technology. Also, peptide hormones in vivo areknown to be expressed in the form of a precursor protein, which then iscleaved by a proteolytic enzyme etc., and there are many reports that aprohormone convertase (or proprotein convertase: PC) is involved in theactivity. In the human full-length nesfatin polypeptide set forth in SEQID NO: 2, the mouse precursor nesfatin polypeptide set forth in SEQ IDNO: 5 and the rat precursor nesfatin polypeptide set forth in 8, thereis a common site (see Working Example 10) that is likely to be cleavedby a subtype of the prohormone convertase, PC1/3 (EC 3.4.21.93, Seidahiet al., DNA and Cell Biology, USA, Vol. 9, 1990, pp. 415-424) or PC2 (EC3.4.21.94, Seidah et al., DNA and Cell Biology, USA, Vol. 9, 1990, pp.415-424), and a possibility was demonstrated that nesfatin-1polypeptides set forth in SEQ ID NO: 13 to SEQ ID NO: 15, respectively,are excised.

Also, when a nesfatin (Mut) having a mutation at the site cleaved withprohormone convertase was given to the ventricle of the rat brain, anunexpected result was obtained that the effect of suppressing foodintake could not be noted (Working Example 14). Therefore, thissuggested a possibility that the nesfatin-1 polypeptide is a functionalmolecule involved in food intake control and/or body weight the controlin a living body, and that in order for the nesfatin polypeptide to befunctional, the process of being processed by a protease such asprohormone convertase is important.

From the foregoing, the fact was not known at all that nesfatin/NEFAfunctions as a hormone precursor like proinsulin, and thus it was foundfor the first time in the present invention after the expression sitefor nesfatin/NEFA and the expression of nesfatin/NEFA in cells thatexpress PC1/3 and PC2 were analyzed and the activity and structure wereintensively studied. Also, since there are secretary proteins that arenot processed by PC1/3 and PC2 despite the presence of sequences Arg-Argor Lys-Arg that are recognition sites of the prohormone convertase, itcannot be easily inferred that nesfatin-1 is excised from nesfatin/NEFAand exhibits the activity of suppressing food intake and/or suppressingbody weight gain.

From the foregoing, the present invention also relates to nesfatin-1polypeptides set forth in SEQ ID NOs: 13-15. As described above, saidnesfatin-1 polypeptides have an activity of suppressing food intakeand/or suppressing body weight gain. The amino acid sequence of mousenesfatin-1 polypeptide is shown in SEQ ID NO: 14. A nesfatin polypeptidehaving such a sequence can be obtained by cleaving the nesfatinpolypeptide set forth in SEQ ID NO: 14 with the prohormone convertasefollowed by purification with a technique such as reverse phasechromatography or by performing the step of binding to and releasingfrom an antibody against the nesfatin-1 polypeptide described below.

Furthermore, after continued intensive investigation on the structure ofthe nesfatin-1 polypeptide and the activity of suppressing food intakeand/or suppressing body weight gain, it was found that a novelpolypeptide comprising 30 amino acids having a sequence corresponding toamino acids 24 to 53 of the nesfatin polypeptide set forth in SEQ ID NO:14 exhibits an activity of suppressing food-intake and/or suppressingbody weight gain (illustrated in Working Example 20), and saidpolypeptide was named nesfatin-1M30 (SEQ ID NO: 41). The discovery ofthe nesfatin-1M30 polypeptide indicates that when a polypeptide thatcontains the part corresponding to nesfatin-1M30 is present even afterthe nesfatin polypeptide or the nesfatin-1 polypeptide wasphysiologically or artificially cleaved or digested, said polypeptideretains an activity of suppressing food intake and/or suppressing bodyweight gain.

Furthermore, for the structure of nesfatin-1M30 comprising 30 aminoacids, the activity of the site having an activity of suppressing foodintake was investigated. The result indicated that nesfatin-1M16comprising 16 amino acids which is a partial peptide of its peptide,nesfatin-1M14 comprising 14 amino acids, and nesfatin-1M10M comprising10 amino acids have an activity of suppressing food intake and/orsuppressing body weight gain (Working Example 22).

When the sequences of nesfatin-1 comprising 82 amino acids set forth inSEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 was analyzed on homologywith the amino acid sequence of a factor having an activity ofcontrolling food intake, no sequences having a high homology were found,and thus it was impossible to estimate that these nesfatin-1M30,nesfatin-1M16, nesfatin-1M14 and nesfatin-1M10M have an activity ofsuppressing food intake and/or suppressing body weight gain based on theconventional technology. It was also demonstrated that thesenesfatin-1M30, nesfatin-1M16, nesfatin-1M14 and nesfatin-1M10M areactive even if they are derived from inactive human-derived nesfatin ornesfatin-1.

Thus, the present invention relates to the nesfatin-1M30 polypeptide setforth in SEQ ID NOs: 39-41, the nesfatin-1M16 polypeptide set forth inSEQ ID NO: 65, 68 or 71, the nesfatin-1M14 polypeptide set forth in SEQID NO: 66, 69 or 72, or the nesfatin-1M10M polypeptide set forth in SEQID NO: 68, 70 or 73. As described above, said nesfatin-1M30 polypeptide,nesfatin-1M16 polypeptide, nesfatin-1M14 polypeptide and nesfatin-1M10Mpolypeptide have an activity of suppressing food intake and/orsuppressing body weight gain. Also, the amino acid sequence of humannesfatin-1M30 polypeptide is shown in SEQ ID NO: 39. Also, thepolypeptides comprising an amino acid sequence set forth in SEQ ID NO:39, SEQ ID NO: 40 or SEQ ID NO: 41 excluding the polypeptides comprisingan amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ IDNO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are included in thenesfatin-1M30 polypeptide. As examples of such polypeptides, there canbe mentioned those polypeptides that were physiologically orartificially cleaved or digested from the nesfatin polypeptide or thenesfatin-1 polypeptide and that contain a sequence corresponding tonesfatin-1M30.

With regard to Nucleobindin I (NUCB1) that has a high homology withNEFA/nesfatin in terms of the amino acid sequence and the nucleotidesequence of the gene and that belongs to the same family, it wasinvestigated whether NUCB1-M30 which is a site corresponding tonesfatin-1M30 of NUCB1 exhibits a similar activity. The result revealedthat NUCB1-M30 also has an activity of suppressing food intake and/orsuppressing body weight gain (Working Example 23). When human, rat andmouse nesfatins were compared, it was found, the amino acid sequence hasbeen highly conserved at the site corresponding to nesfatin-1 ofnesfatin and NUCB1 of each species, specifically at the sitecorresponding to nesfatin-1M30. From the foregoing, it is inferred thatNUCB1-M16 comprising 16 amino acids of NUCB1, NUCB1-M14 comprising 14amino acids and NUCB1-M10M comprising 10 amino acids of NUCB1 also havean activity of suppressing food intake and/or suppressing body weightgain similarly to the nesfatin-1M16 polypeptide, the nesfatin-1M14polypeptide and the nesfatin-1M10M polypeptide.

Thus, the present invention relates to the NUCB1-M30 polypeptide setforth in SEQ ID NOs: 101-103, the NUCB1-M16 polypeptide set forth in SEQID NO: 107, 110 or 113, the NUCB1-M14 polypeptide set forth in SEQ IDNO: 108, 111 or 114, or the NUCB1-M10M polypeptide set forth in SEQ IDNO: 109, 112 or 115. Said NUCB1-M30 polypeptide, NUCB1-M16 polypeptide,NUCB1-M14 polypeptide and NUCB1-M 10M polypeptide also have an activityof suppressing food intake and/or suppressing body weight gain asdescribed above.

The present invention also relates to a polypeptide that has a homologyof at least 60% with any of the amino acid sequences set forth in SEQ IDNOs: 13-15, 39-41, 65-73, 101-103 and 107-115 and that has an activityof suppressing food intake and/or suppressing body weight gain. Thehomology with the amino acid sequence set forth in SEQ ID NOs: 13-15,39-41, 65-73, 101-103 and 107-115 is preferably 70% or greater and morepreferably 80% or greater. A representative example thereof includes thenesfatin-1M30 polypeptide of a non-human animal. For example, as apolypeptide that has a homology of 60% or greater with the amino acidsequence of the human nesfatin-1M30 polypeptide (SEQ ID NO: 39) and thathas an activity of suppressing food intake and/or suppressing bodyweight gain, there can be mentioned, but not limited to, a mousenesfatin-1M30 polypeptide (SEQ ID NO: 41) and a rat nesfatin-1M30polypeptide (SEQ ID NO: 40).

Among the polypeptides comprising an amino acid sequence that has ahomology of at least 60% with any of the amino acid sequences set forthin SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 and 107-115, the selectionof a polypeptide having an activity of suppressing food intake and/orsuppressing body weight gain may be performed by introducing saidpolypeptide or a nucleic acid molecule encoding said polypeptide locallyor systemically into a test animal, and then selecting a polypeptidethat suppresses the amount of food intake and/or the body weight of saidanimal. In an alternative method, the selection may be performed byintroducing an antibody against said polypeptide or an antisenseoligonucleotide molecule or a RNAi molecule that can suppress theexpression of a gene encoding said polypeptide locally or systemicallyinto a test animal, and then selecting a polypeptide that suppresses theamount of food intake and/or the body weight of said animal. Such apolypeptide is referred to hereinafter as altered nesfatin-1M30, alterednesfatin-1M16, altered nesfatin-1M14, altered nesfatin-1M10M, alteredNUCB1-M30, altered NUCB1-M16, altered NUCB1-M14 and altered NUCB1-M10M.

Furthermore, the present invention also relates to a polypeptide thatcomprises an amino acid sequence in which some of the amino acids havebeen deleted, inserted or substituted in any of the amino acid sequencesset forth in SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 and 107-115 andthat has an activity of suppressing food intake and/or suppressing bodyweight gain. Such a polypeptide can be obtained by replacing one or moreamino acid residues with amino acids that are chemically or structurallysimilar to said amino acids in an amino acid sequence set forth in, forexample, SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or 107-115. Specificembodiments of the substitution of amino acids that are chemically orstructurally similar, i.e. the substitution of highly conserved aminoacids, are well known to a person skilled in the art. For example,chemically or structurally, glycine (Gly) is similar to proline (Pro),alanine (Ala) and valine (Val), leucine (Leu) is similar to isoleucine(Ile), glutamic acid (Glu) is similar to glutamine (Gln), aspartic acid(Asp) is similar to asparagine (Asn), cysteine (Cys) is similar tothreonine (Thr), Thr is similar to serine (Ser) and Ala, and lysine(Lys) is similar to arginine (Arg). Further, as alternative method, aperson skilled in the art can easily refer to a amino acid matrixmethod, which represents in what extent can be made substitution of aamino acid, as a matrix, such as PAM (Wilbur, Molecular Biology andEvolution) (USA), 1985, Vol. 2, pp. 434-447), BLOSUM (Henikoff et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica) (USA) 1992, Vol. 89, pp. 10915-10919, and easily substitute anamino acid, considering height of its score.

Also, among the polypeptides comprising an amino acid sequence in whichsome of the amino acids have been deleted, inserted or substituted inany of the amino acid sequences set forth in SEQ ID NOs: 13-15, 39-41,65-73, 101-103 and 107-115, the selection of a polypeptide that has anactivity of suppressing food intake and/or suppressing body weight gaincan be performed in a manner similar to that described in the selectionof the above modified nesfatin. Hereinbelow, such a polypeptide is alsoreferred to as altered nesfatin-1M30.

Furthermore, the present invention also relates to a polypeptide thatcomprises an amino acid sequence having a homology of at least 60% withany of the amino acid sequences set forth in SEQ ID NOs: 3, 6 and 9; oran amino acid sequence in which some of the amino acids have beendeleted, inserted or substituted in any of the amino acid sequences setforth in SEQ ID NOs: 3, 6 and 9, and that has an activity of suppressingfood intake and/or suppressing body weight gain. Similarly to theabove-mentioned altered products, said polypeptide is also referred toas an altered nesfatin. Said altered nesfatin, similarly to theabove-mentioned altered nesfatin-1M30 etc., preferably has a homology of70% or greater and more preferably 80% or greater with the amino acidsequence set forth in SEQ ID NOs: 3, 6 or 9.

As described above, it was demonstrated by the present invention thatfor a nesfatin polypeptide to be functional, the step of its beingprocessed by a protease contained in the living body such as prohormoneconvertase is important. For that purpose, said altered nesfatinpreferably is a polypeptide that produces in the living body nesfatin-1,nesfatin-1M30, nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30,NUCB1-M16, NUCB1-M14, or NUCB1-M10M (a polypeptide comprising an aminoacid sequence set forth in SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or107-115), or an altered product thereof. It is necessary for such analtered nesfatin to have a recognition site, in its amino acid sequence,for a cleaving enzyme such as a protease contained in the living body.As such a cleaving enzyme, there can be mentioned for example prohormoneconvertase (proprotein convertase: PC), and as such a prohormoneconvertase, there can be mentioned for example furin, PC1 (also known asPC3), PC2, PACE4, PC4, PC6 (also known as PC5) and LPC (also known asPC7 or PC8) (The FASEB Journal, 1216, vol. 17, July 2003). As long asthe protease produces nesfatin-1 etc. in the periphery or the ventricleof the brain, the type of the protease is not specifically limited.

For similar reasons, the position of the recognition site for saidcleaving enzyme is not specifically limited. However, based on theexperimental results on mouse-derived nesfatin-1, nesfatin-2, nesfatin-3and nesfatin-2/3 (Working Example 10), altered nesfatin is preferablyestablished so as to contain at least one recognition site for acleaving enzyme contained in the living body in between the amino acids82 and 83 (a recognition site for prohormone convertase that producesnesfatin-1) of SEQ ID NO: 3, 6 or 9 and between the amino acids 163 and164 (another recognition site for prohormone convertase) of SEQ ID NO:3, 6 or 9, i.e. in an amino acid sequence corresponding to the aminoacid numbers 82-162 of SEQ ID NO: 3, 6 or 9, in that it does not affectthe activity of the resulting polypeptide.

Also, the confirmation of whether said altered product have an activityof suppressing food intake and/or suppressing body weight gain includingan activity of being cleaved and becoming active in vivo can be made, asfor the selection of the above-mentioned altered nesfatin-1M30 etc., byintroducing said polypeptide or a nucleic acid molecule encoding saidpolypeptide locally or systemically into a test animal, and thenselecting a polypeptide that suppresses the amount of food intake and/orthe body weight of said animal. In an alternative method, the selectionmay be performed by introducing an antibody against said polypeptide oran antisense oligonucleotide molecule or a RNAi molecule that cansuppress the expression of a gene encoding said polypeptide locally orsystemically into a test animal, and then selecting a polypeptide thatsuppresses the amount of food intake and/or the body weight of saidanimal.

The nesfatin polypeptide, nesfatin-1 polypeptide, nesfatin-1M30,nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16,NUCB1-M14, or NUCB1-M10M of the present invention include those in whichat least one amino acid has been added to the N terminal or C terminalthereof. Such nesfatin polypeptides include those in which a methionineresidue, an acetyl residue or a pyroglutamic acid residue or the likehas been added to the N terminal of the amino acid sequence set forth inSEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9, and those in which asuitable tag sequence (typically a histidine tag or a FLAG tag) has beenadded to the N terminal or the C terminal. Nesfatin polypeptides etc.having such construction have an advantage that they can be easilypurified using a metal chelate carrier or antibody. Also, when nesfatinhas been processed in the living body by prohormone convertase, it isthought, a polypeptide in which the recognition site for prohormoneconvertase has been added to the C terminal of nesfatin-1 is produced,and such a polypeptide is also encompassed by the present invention.

Also, the nesfatin polypeptide, the nesfatin-1 polypeptide,nesfatin-1M30, nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30,NUCB1-M16, NUCB1-M14, or NUCB1-M10M of the present invention includethose in which at least one amino acid residue has been modified by acompound or a peptide. Such a nesfatin polypeptide includes, in additionto the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9,polypeptides that are obtained by enzymatically or chemicallyassociating a peptide other than the nesfatin polypeptide or afluorescent substance etc. by a known method (Hermanson et al.,Bioconjugate Techniques, USA, 1996, issued by Academic Press), andincludes, for example, one in which the amino acid sequence of anAequorea-derived fluorescence protein or a secretary alkalinephosphatase has been added (so-called fusion protein). The presence ofthese fusion proteins can be easily detected; for example, bydetermining fluorescence intensity for a fusion protein with anAequorea-derived fluorescence protein, or by determining the intensityof color development, emission or fluorescence resulting from thereaction with said enzyme and its substrate for a fusion protein with asecretary alkaline phosphatase. For such a nesfatin polypeptide, aneffect on food intake control and/or body weight control can beidentified by investigating the activity by a method described inWOrking Example 6 etc.

The present invention relates to a pharmaceutical composition forsuppressing food intake and/or suppressing body weight gain, saidcomposition comprising as an active ingredient any of the nesfatinpolypeptide, the nesfatin-1 polypeptide, nesfatin-1M30, nesfatin-1M16,nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16, NUCB1-M14, orNUCB1-M10M, or altered products thereof (hereinafter referred tocollectively as “the nesfatin polypeptide etc.”), or a peptidecomprising some of the amino acid sequences of said nesfatin polypeptideetc. The peptide comprising some of the amino acid sequence of saidnesfatin polypeptide refers to a peptide in which some of the amino acidsequence of said polypeptide has been deleted within the extent ofretaining the activity of suppressing food intake and/or suppressingbody weight gain.

<A Pharmaceutical Composition for Treating or Preventing a Disease forwhich Enhanced Food Intake and Body Weight Gain is a Problem>

The present invention relates to a pharmaceutical composition fortreating or preventing a disease for which enhanced food intake and bodyweight gain is a problem, said composition comprising, as an activeingredient, any of the above nesfatin polypeptide etc., or a peptidecomprising some of the amino acid sequence of said polypeptide.

A pharmacological effect of the nesfatin polypeptide etc. of the presentinvention is demonstrated by the results of the administration ofnesfatin polypeptide to the third ventricle of the rat brain (WorkingExamples 6 and 25), the administration of nesfatin-1 polypeptide intothe third ventricle of the rat brain (Working Examples 12, 13, 27 and34), the administration of nesfatin-1 polypeptide into the abdominalcavity or the subcutaneous of mouse (Working Examples 18 and 19), theadministration of nesfatin-1M30 polypeptide to the abdominal cavity ofmouse (Working Example 20). A pharmacological effect in pathologicalmodel animals is illustrated by the administration of nesfatin-1polypeptide to the third ventricle of the brain in a model animalindicating leptin resistance, i.e., Zucker (fa/fa) rat (Working Example17), and a pharmacological effect on pathology of leptin resistance,which is also a problem in human adiposis, is illustrated. Further, itis pharmacologically illustrated that food intake may be controlledthrough the mechanism different from melanocortine system, which is aknown factor related to the control of food intake, by theadministration experiment in Agouti yellow mouse (Working Example 18).By the illustrated facts, it is demonstrated that nesfatin polypeptideetc. may be used as a pharmaceutical composition for treating orpreventing a disease for which enhanced food intake and body weight gainis a problem.

The diseases for which enhanced food intake and body weight gain is aproblem include, for example, obesity, diabetes mellitus, hypertension,hyperlipidemia, hyperuricemia, fatty liver, cardiac diseases, cerebralvascular diseases, sleep apnea syndrome, orthopedic diseases, menstrualdisorders and malignant tumors. Obesity includes adiposis, apathological condition that requires body weight reduction when a healthproblem associated with obesity is complicated or its complication isclinically predicted. Orthopedic diseases include osteoarthritis due tooverweight, lumber disorders (spondylosis deformans), low back pain(acute low back pain) etc. Also malignant tumors include breast cancer,uterine cancer, colon cancer, kidney cancer, esophageal cancer,pancreatic cancer, liver cancer and gallbladder cancer.

The pharmaceutical composition of the present invention may contain anypharmaceutically acceptable additives. Formulations usingpharmaceutically acceptable additives may be prepared by a methoddescribed in “Remington: The Science and Practice of Pharmacy, 20thedition, University of the Sciences in Philadelphia, Williams & Wilkins,issued on Dec. 15, 2000”. One dosage form of such a pharmaceuticalcomposition is presented as a liquid prepared by dissolving, suspendingor emulsifying them in an aqueous or oleaginous solution. Such solventsused include, for example, distilled water, physiological saline etc.for injection as an aqueous liquid, and in addition, an osmoregulatoryagent (for example, D-glucose, D-sorbitol, D-mannitol, and sodiumchloride), a suitable solubilizing agent such as alcohols (for example,ethanol), polyalcohols (for example, propylene glycol, and polyethyleneglycol), nonionic surfactants (for example, polysorbate 80,polyoxyethylenated hydrogenated castor oil 50) and the like may be used.Also, as the solvents, an oleaginous solution may be used, and saidoleaginous solution includes sesame oil, soybean oil etc. and, as thesolubilizing agent, benzyl benzoate, benzyl alcohol etc. may be used incombination. In such liquids, there can be used, as appropriate,additives such as buffers (such as phosphate buffers and acetatebuffers), soothing agents (such as benzalkonium chloride and procainehydrochloride), stabilizers (such as human serum albumin andpolyethylene glycol), preservatives (such as ascorbic acid, erythorbicacid, and salts thereof), colorants (such as copper chlorophyll,β-carotene, Red No. 2, and Blue No. 1), preservatives (such as ascorbicacid, erythorbic acid, and salts thereof), antiseptics (such asparaoxybenzoate ester, phenol, benzethonium chloride and benzalkoniumchloride), thickeners (such as hydroxypropyl cellulose, carboxymethylcellulose, and salts thereof), stabilizers (such as human serum albumin,mannitol and sorbitol), and corrigents (such as menthol and citrusflagrances). Another dosage form of pharmaceutical compositions (inorder to unify the expression with the above several “pharmaceuticalcompositions”) includes solid forms such as powders, tablets, granules,capsules, pills, suppositories, and lozenges. In the case of solid formsthat are administered in the form of oral preparations, additives usedinclude excipients (such as crystalline cellulose, lactose and starch),lubricants (such as magnesium stearate and talc), binders (such ashydroxypropyl cellulose, hydroxypropyl methyl cellulose and macrogol),disintegrants (such as starch and carboxymethyl cellulose calcium), andthe like. Also, as needed, there can be used antiseptics (such as benzylalcohol, chlorobutanol, methyl paraoxybenzoate and propylparaoxybenzoate), antioxidants, colorants, sweeteners and the like.Furthermore, another form also includes pharmaceutical compositions forapplication to mucosa, and for this form of formulations, in order toimpart adsoptivity to mucosa and retentivity, there can be containedadditives such as tackifiers, tackiness enhancers, thickener, thickeningagent (such as mucin, agar, gelatin, pectin, carrageenan, sodiumalginate, locust bean gum, xanthan gum, tragacanth gum, gum arabic,chitosan, pullulan, waxy starch, sucralfate, cellulose and itsderivatives (such as hydroxypropyl methyl cellulose, polyglycerin fattyacid esters, acrylic-(meth)acrylic alkyl copolymers or salts thereof,and polyglycerin fatty acid esters). However, dosage forms ofpharmaceutical compositions and solvents and additives to be deliveredto the living body are not limited to the above, and can be selected, asappropriate, by a person skilled in the art.

For the purpose of ameliorating disease conditions, the abovepharmaceutical composition can be administered orally or parenterally.In the case of oral administration, dosage forms such as granules,powders, tablets, capsules, liquids, syrups, emulsions or suspensions,and elixirs can be selected. In the case of parenteral administration,it can be transnasal agents, and liquids, suspensions, solidformulations can be selected. Other agents in the form of parenteraladministration, it can be in the form of injections, and injectionsselected may be hypodermic injections, intravenous injections, dripinjections, intramuscular injections, intraventricular injections, orintraperitoneal injections and the like. Other formulations for use inparenteral administration include, for example, transmucosalpreparations other than suppositories, sublingual tablets, transdermalpreparations, nasal preparations, and the like. Furthermore,intravascular local administration can be performed, in embodimentswhere they are involved in or applied to stents or anti-intravascularlocal embolism agents.

The dosage of the above pharmaceutical composition may vary depending onthe age, sex, body weight of the patient, condition, therapeutic effect,administration regimen, treatment period, or the types of activeingredients contained in said pharmaceutical composition, but its onedosage is usually in the range of 0.1-500 mg per person for adults, andpreferably in the range of 0.5 mg-20 mg. The dosage may vary dependingon various conditions, and thus a dosage smaller than that describedabove may sometimes be sufficient, or at other times a dosage greaterthan the above may be required.

Also, gene encoding the nesfatin polypeptide or nesfatin-1 polypeptidemay be used in embodiments of gene therapy. Said gene therapy, forexample, can attain the therapeutic effect by introducing said gene intothe living body. Techniques for introducing a gene encoding a proteinthat provides therapeutic effect and allowing it to be expressed inorder to treat a disease is known (Kaneda, Folia PharmacologicaJaponica, 2001, Vol. 117, pp. 299-306).

Furthermore, by administering a transformant in the form in which a genecontaining a nesfatin polypeptide or a nesfatin-1 polypeptide has beenintroduced and that expresses said polypeptide, preferably byadministering a transformant that employs a host transplantable into aspecies into which the gene is to be introduced, treatment with thenesfatin polypeptide or nesfatin-1 polypeptide produced by saidtransformant can be performed.

<A Nucleic Acid Molecule Encoding a Polypeptide Having an Activity ofSuppressing Food Intake and/or Suppressing Body Weight Gain>

The present invention also relates to a nucleic acid molecule encodingany of the above nesfatin polypeptide etc. As the nucleic acid moleculeencoding the above nesfatin polypeptide, there can be mentioned anucleic acid molecule (SEQ ID NO: 1) comprising the nucleotide sequenceof a gene encoding the human precursor nesfatin polypeptide, a nucleicacid molecule (SEQ ID NO: 4) comprising the nucleotide sequence of agene encoding the mouse precursor nesfatin polypeptide, a nucleic acidmolecule (SEQ ID NO: 7) comprising the nucleotide sequence of a geneencoding the rat precursor nesfatin polypeptide, a nucleic acid molecule(SEQ ID NO: 10) comprising the nucleotide sequence of a gene encoding amatured nesfatin polypeptide in which a signal peptide portion has beenremoved, a nucleic acid molecule (SEQ ID NO: 11) comprising thenucleotide sequence of a gene encoding the mouse matured nesfatinpolypeptide, a nucleic acid molecule (SEQ ID NO: 12) comprising thenucleotide sequence of a gene encoding the rat matured nesfatinpolypeptide, and the like.

As nucleic acid molecules encoding the above nesfatin-1 polypeptides,there can be mentioned a nucleic acid molecule (SEQ ID NO: 18) encodingthe human nesfatin-1 polypeptide, a nucleic acid molecule (SEQ ID NO:19) encoding the mouse nesfatin-1 polypeptide, a nucleic acid molecule(SEQ ID NO: 20) encoding the rat nesfatin-1 polypeptide, and the like.

As nucleic acid molecules encoding the above nesfatin-1M30 polypeptides,there can be mentioned a nucleic acid molecule (SEQ ID NO: 44) encodingthe human nesfatin-1M30 polypeptide, a nucleic acid molecule (SEQ ID NO:46) encoding the mouse nesfatin-1M30 polypeptide, a nucleic acidmolecule (SEQ ID NO: 45) encoding the rat nesfatin-1M30 polypeptide, andthe like.

As nucleic acid molecules encoding the nesfatin-1M16 polypeptides, therecan be mentioned a nucleic acid molecule (SEQ ID NO: 74) encoding thehuman nesfatin-1M16 polypeptide, a nucleic acid molecule (SEQ ID NO: 80)encoding the mouse nesfatin-1M16 polypeptide, a nucleic acid molecule(SEQ ID NO: 77) encoding the rat nesfatin-1M16 polypeptide, and thelike.

As nucleic acid molecules encoding the nesfatin-1M14 polypeptides, therecan be mentioned a nucleic acid molecule (SEQ ID NO: 75) encoding thehuman nesfatin-1M14 polypeptide, a nucleic acid molecule (SEQ ID NO: 81)encoding the mouse nesfatin-1M14 polypeptide, a nucleic acid molecule(SEQ ID NO: 78) encoding the rat nesfatin-1M14 polypeptide, and thelike.

As nucleic acid molecules encoding the above nesfatin-1M10Mpolypeptides, there can be mentioned a nucleic acid molecule (SEQ ID NO:76) encoding the human nesfatin-1M10M polypeptide, a nucleic acidmolecule (SEQ ID NO: 82) encoding the mouse nesfatin-1M10M polypeptide,a nucleic acid molecule (SEQ ID NO: 79) encoding the rat nesfatin-1M10Mpolypeptide, and the like.

As nucleic acid molecules encoding the NUCB1-M30 polypeptides, there canbe mentioned a nucleic acid molecule (SEQ ID NO: 104) encoding the humanNUCB1-M30 polypeptide, a nucleic acid molecule (SEQ ID NO: 106) encodingthe mouse NUCB1-M30 polypeptide, a nucleic acid molecule (SEQ ID NO:105) encoding the rat NUCB1-M30 polypeptide, and the like.

As nucleic acid molecules encoding the NUCB1-M16 polypeptides, there canbe mentioned a nucleic acid molecule (SEQ ID NO: 116) encoding the humanNUCB1-M16 polypeptide, a nucleic acid molecule (SEQ ID NO: 122) encodingthe mouse NUCB1-M16 polypeptide, a nucleic acid molecule (SEQ ID NO:119) encoding the rat NUCB1-M16 polypeptide, and the like.

As nucleic acid molecules encoding the NUCB1-M14 polypeptides, there canbe mentioned a nucleic acid molecule (SEQ ID NO: 117) encoding the humanNUCB1-M14 polypeptide, a nucleic acid molecule (SEQ ID NO: 123) encodingthe mouse NUCB1-M14 polypeptide, a nucleic acid molecule (SEQ ID NO:120) encoding the rat NUCB1-M14 polypeptide, and the like.

As nucleic acid molecules encoding the above NUCB1-M10M polypeptides,there can be mentioned a nucleic acid molecule (SEQ ID NO: 118) encodingthe human NUCB1-M10M polypeptide, a nucleic acid molecule (SEQ ID NO:124) encoding the mouse NUCB1-M10M polypeptide, a nucleic acid molecule(SEQ ID NO: 121) encoding the rat NUCB1-M10M polypeptide, and the like.

Also, the nesfatin polypeptides etc. of the present invention include,as described above, those in which at least one amino acid has beenadded to the N terminal or the C terminal, and those in which at leastone amino acid residue has been modified by a compound or a peptide. Asnucleic acid molecules encoding such nesfatin polypeptides etc., in thecase of the nesfatin polypeptide for example, a nucleic acid moleculeencoding a polypeptide in which a polypeptide has been added to the Nterminal of the sequence of SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9can be obtained by adding a nucleotide sequence having the nucleotidesequence ATG and a codon encoding an amino acid sequence desired to beadded behind it to the 5′-end of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ IDNO: 12. Also, a nucleic acid molecule encoding a polypeptide in which arecognition (cleaving) sequence of protease such as prohormoneconvertase has been added to the N terminal and/or the C terminal of theamino acid sequence of the nesfatin polypeptide etc. is encompassed bythe present invention, but it is not limited to them.

In addition, a nucleic acid molecule is included that comprises anucleotide sequence in which a gene sequence encoding anAequorea-derived fluorescence protein or a secretary alkalinephosphatase has been added to the 5′- or 3′-end of the gene sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 in such a form that the aminoacid sequence of each protein can be translated.

Nucleic acid molecules that encode the above altered nesfatin etc.include nucleic acid molecules encoding a polypeptide that comprises anamino acid sequence having a homology of at least 60% with any of theamino acid sequence set forth in SEQ ID NOs: 3, 6, 9, 13-15, 39-41,65-73, 101-103 or 107-115 and that has an activity of suppressing foodintake and/or suppressing body weight gain, and preferably said homologyis 70% or greater and more preferably 80% or greater. As therepresentative example thereof, there can be mentioned, in the case ofthe nesfatin-1M30 polypeptide for example, a nesfatin-1M30 polypeptideof a non-human animal species. For example, nucleic acid moleculesencoding a polypeptide that comprises an amino acid sequence having ahomology of 60% or greater with the amino acid sequence (SEQ ID NO: 39)of human nesfatin-1M30 polypeptide etc. and that has an activity ofsuppressing food intake and/or suppressing body weight gain include anucleic acid molecule (SEQ ID NO: 46) encoding the mouse nesfatin-1M30polypeptide, a nucleic acid molecule (SEQ ID NO: 45) encoding the ratnesfatin polypeptide, a nucleic acid molecule (SEQ ID NO: 104) encodingthe human NUCB1-M30 polypeptide, a nucleic acid molecule (SEQ ID NO:106) encoding the mouse NUCB1-M30 polypeptide, and a nucleic acidmolecule (SEQ ID NO: 105) encoding the rat NUCB1-M30 polypeptide and thelike, but not limited to these nucleic acid molecules.

Also, nucleic acid molecules encoding the above altered nesfatin etc.include those nucleic acid molecules encoding a polypeptide comprisingan amino acid sequence in which some of the amino acids have beendeleted, inserted or substituted in any of the amino acid sequence setforth in SEQ ID NOs: 6, 9, 13-15, 39-41, 65-73, 101-103 or 107-115 andthat has an activity of suppressing food intake and/or suppressing bodyweight gain.

Furthermore, the present invention also relates to a nucleic acidmolecule that hybridizes to a nucleotide sequence set forth in SEQ IDNOs: 4, 7, 11, 12, 18, 19, 20, 44-46, 74-82, 104-106 or 116-124, or apartial sequence thereof under a stringent condition, and that encodes apolypeptide having an activity of suppressing food intake and/orsuppressing body weight gain. Such a nucleic acid molecule can beobtained by a hybridization method that employs a nucleotide sequenceset forth in SEQ ID NOs: 4, 7, 10, 11, 12, 18, 19, 20, 44-46, 74-82,104-106 or 116-124, or a partial sequence thereof. Specifically, alibrary is constructed in which a plasmid vector or a phage vectorhaving inserted therein a cDNA or genomic DNA fragment derived from anytype of organism has been introduced into a host such as Escherichiacoli (E. coli), and then the library is cultured on an agar medium platecontaining a suitable selection drug. Then, the resulting recombinant E.coli clone or a phage clone is transferred to a nitrocellulose membraneetc., and then the cells or phage are lyzed in an alkali- orsurfactant-containing condition so as to immobilize DNA containedtherein onto the membrane. To the membrane is reacted a suitablehybridization solution at a suitable temperature in which a linearizedprobe obtained by labelling DNA comprising a nucleotide sequence setforth in SEQ ID NOs: 4, 7, 10, 11, 12, 18, 19, 20, 44-46, 74-82, 104-106or 116-124, or a partial sequence thereof with ³²P has been dissolved.After the reaction, the membrane is washed with ×2 SSC to remove theexcessive probe, and then washed under a highly stringent condition suchas ×0.1 SSC at 65° C., or a medium stringent condition such as ×0.5 SSCat 65° C., and then the membrane is contacted with an X-ray film in thedark for exposure. After an X-ray film that was exposed in a deepfreezer for a few hours to a few days is developed to detect the exposedspots, and E. coli or phage clones located at positions corresponding tothe original plate from which transfer was made to the membrane areharvested and cultured, the sequence of the genes that have beeninserted to the vector is analyzed to obtain genes that are highlyhomologous to the nesfatin gene, the nesfatin-1 gene or thenesfatin-1M30 gene. Also, by obtaining clones by hybridization of cDNAor a genome library using the gene sequence or identifying thesurrounding sequences by the primer extension method etc., the genestructure encoding the protein can be elucidated to obtain nucleic acidmolecules homologous to nesfatin, nesfatin-1, nesfatin-1M30,nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16,NUCB1-M14 or NUCB1-M10M. Furthermore, among the nucleic acid moleculesthat hybridize under a stringent condition to the nucleotide sequenceset forth in SEQ ID NOs: 4, 7, 10, 11, 12, 18, 19, 20, 44-46, 74-82,104-106 or 116-124 or the partial sequence of said nucleotide sequence,the nucleic acid molecule encoding a polypeptide having an activity ofsuppressing food intake and/or suppressing body weight gain can beselected by introducing a polypeptide encoded by said nucleic acidmolecule or said nucleic acid molecule locally or systemically to a testanimal and by selecting a nucleic acid molecule that suppresses foodintake and/or suppresses body weight. In alternative method, it can beperformed by introducing an antibody that binds to the polypeptideencoded by said nucleic acid molecule or an antisense oligonucleotidemolecule or a RNAi molecule that can suppresses the expression of thegene encoded by said nucleic acid molecule locally or systemically intoa test animal, and then selecting a nucleic acid molecule thatsuppresses the amount of food intake by and/or the body weight of saidanimal.

<Vector>

The present invention also relates to a vector comprising a nucleic acidmolecule encoding any of the above nesfatin polypeptide etc. Byconstructing a recombinant which is a host cell such as a microorganismhaving introduced therein the nesfatin gene etc. using a vectorcomprising a nucleic acid molecule encoding such nesfatin polypeptideetc., said gene can be stably conserved or replicated. The nucleic acidmolecule of said gene encoding the nesfatin polypeptide integrated intoa vector having a function of being replicated in a host cell can beprepared in large quantities by culturing a recombinant cell in asuitable medium, by cellular growth or by amplifying the copy number ofthe introduced gene in the cell.

Said nucleic acid molecule may be operably linked under the control of aregulatory nucleic acid molecule that controls the expression of saidnucleic acid molecule. As the regulatory nucleic acid molecule thatcontrols the expression of said nucleic acid molecule, there can bementioned a nucleic acid molecule encoding a regulatory sequence forexpressing the integrated gene in the host cell such as a promotersequence or an enhancer sequence, which can be selected as appropriateby a person skilled in the art. By integrating a nucleic acid moleculeencoding the nesfatin polypeptide under the control of a nucleic acidmolecule encoding such a regulatory sequence, the nesfatin polypeptideetc. can be produced in large quantities in any host cell (such as amicrobial cell, a mammalian cell, an insect cell etc.).

Also as the vector that can be used in the present invention, there canbe mentioned a vector that has a promoter upstream to the gene to beexpressed, and a polyadenylation site, transcription terminationsequence etc. downstream thereto. As such an expression vector forvertebrates, there can be mentioned pSV2dhfr (Mol. Cell. Biol., 854,1981) having the SV40 early promoter, pcDNA3.1(+) (Invitrogen Corp.),and pCAGGS (Gene 108:193-200, 1991) and the like, but they are notlimiting and can be selected as appropriate by a person skilled in theart. Such a vector can also be introduced into a mammalian cell for thetreatment of suppressed food intake and/or suppressed body weight gain.

When E. coli is used as the host cell, pBR322 and an improved vectorthereof can be used, but they are not limiting, and various knownmicrobial strains lines and vectors can also be used. As the promoter,there can be mentioned, but not limited to, promoters such as E. colilactose (lac), E. coli trp and the like. Said promoters are those thathave already been characterized and known to a person skilled in theart, and can be assembled synthetically or from known plasmids.

Working Example 5 as a specific example demonstrates that by integratinga nucleic acid molecule encoding a nesfatin polypeptide into a vector toconstruct an E. coli recombinant, a nesfatin polypeptide retaining anactivity of suppressing food intake and/or suppressing body weight gaincould be prepared. Also, Working Example 16 demonstrates that byallowing the amino acid sequences described in SEQ ID NO: 13 to 15 asfusion proteins with glutathione S-transferase (GST) etc, to beexpressed. and purifying using the adsorption to and desorption from aglutathione-immobilized carrier followed by cleavage of the GST portion,the nesfatin-1 polypeptide could be produced by a gene recombinanttechnology.

By introducing the vector of the present invention into a mammalian cellusing the technology for gene therapy for the purpose of suppressingfood intake and/or suppressing body weight gain, the therapeutic effectcan be attained. The method of treating diseases by introducing a geneencoding a protein that provides the therapeutic effect, i.e. thenesfatin polypeptide etc., into a mammalian cell and then allowing it tobe expressed is known (Kaneko, Folia Pharmacologica Japonica, issued in2001, Vol. 117, pp. 299-306).

<Transformant>

The present invention also relates to a transformant comprising anucleic acid molecule encoding any of the above nesfatin polypeptideetc. Such a transformant can be obtained by introducing the geneencoding said polypeptide into a host cell and transforming it. Methodsof transformation include biological methods, physical methods, chemicalmethods and the like. As biological methods, there can be mentioned amethod that employs a virus vector, a method that employs a specificreceptor, cell fusion (Sendai virus (HVJ), polyethylene glycol (PEG),electric cell fusion, micronucleate cell fusion (chromosome transfer)),and the like. Physical methods include the microinjection method, anelectroporation method, the method that employs a gene particle gun.Chemical methods include the calcium phosphate precipitation method, theliposome method, the DEAE-dextran method, the protoplast method, theerythrocyte ghost method, the erythrocyte membrane ghost method, themicrocapsule method, which can be selected and performed as appropriateby a person skilled in the art. The transformant obtained can becultured according to a standard method, and the nesfatin polypeptideetc. can be produced. As the media used for culturing, various mediacommonly used can be as appropriate selected depending on the host celladopted, and culturing can also be performed under a condition suitablefor the growth of the host cell.

Various means for expressing the protein of interest in an eukaryoticcell are known per se in said field of art. For example, an expressionsystem in yeast includes “The expression of protein in yeast” describedin Japanese Unexamined Patent Publication (Kokai) No. 57-159489, anexpression system in plant cells includes “An improved method andequipment for introducing a biological substance into a living cell”described in Patent Publication No. 2517813 or “A method of introducinga gene into a plant cell and a plant cell-treatment equipment for geneintroduction” described in Japanese Unexamined Patent Publication(Kokai) No. 2003-274953, an expression system in insect cells includes“A method of producing a recombinant baculovirus expression vector”described in Japanese Unexamined Patent Publication (Kokai) No.60-37988, and an expression system in mammalian cells includes“Improvement in eukaryotic expression” described in Japanese UnexaminedPatent Publication (Kokai) No. 2-171198, but there are many othersystems in addition to these.

As host cells for use in transformation, both eukaryotic host cells andprokaryotic host cells can be used. Eukaryotic host cells includevertebrates, yeast, plant cells, insect cells and the like. As plantcells, there can be mentioned tissue sections of dicotyledons andmonocotyledons, cells isolated from the tissue, cells derived fromcallus formed from the tissue and the like. Vertebrate cells include,for example, CHO cells, 293T cells, COS7 cells and the like. Prokaryotichost cells include, for example, E. coli, Bacillus subtilis,Streptomyces and the like, and as E. coli, Escherichia coli strain K12is often used. When a vertebrate is used as the host cell, thetransformant obtained can be introduced into a mammal for cell therapyof suppressing food intake and/or suppressing body weight gain. As suchtransformants, those that were confirmed to express the gene encodingthe introduced nesfatin polypeptide etc. are preferred. In administeringsaid transformant, it is preferred to administer said transformant bydispersing in various buffers, physiological saline etc. (JapaneseUnexamined Patent Publication (Kokai) No. 2003-342201)

<Antibody>

The present invention relates to antibody that binds to any of the abovenesfatin polypeptide etc. Such an antibody can be obtained by a methodknown to a person skilled in the art. The antibody for use in thepresent invention can be polyclonal antibody or monoclonal antibody(Milstein et al., Nature (England), issued on Oct. 6, 1983, Vol. 305,No. 5934, pp. 537-540). For example, a polyclonal antibody against thenesfatin polypeptide, the nesfatin-1 polypeptide, the nesfatin-1M30polypeptide, nesfatin-1M16, nesfatin-1M14, nesfatin-1M10M, NUCB1-M30,NUCB1-M16, NUCB1-M14 or NUCB1-M10M can be collected from the serum etc.of a mammal sensitized with the antigen. Furthermore, in anotherexample, it can be collected from the serum etc. of a mammal sensitizedwith a peptide having a sequence comprising a partial sequence of thenesfatin polypeptide etc. In a more specific example, as shown inWorking Example 3, an antibody that binds to the nesfatin polypeptideetc. can be obtained by binding a peptide (SEQ ID NO: 24) havingsequence corresponding to amino acids 141-152 of SEQ ID NO: 8 to acarrier protein and using it as the antigen to immunize an animal. Whenadministered to an animal, this antibody exhibits an effect of enhancingfood intake and increasing body weight of said animal (Working Example7). In still another example, a peptide (SEQ ID NO: 32) having asequence corresponding to amino acids 48-62 of SEQ ID NO: 8 is bound toa carrier protein, which is used as the antigen to immunize an animal sothat an antibody that binds to the nesfatin polypeptide etc. can beobtained (Working Example 10). The polypeptide set forth in SEQ ID NO:32 consists of amino acid sequences common to the nesfatin polypeptide,nesfatin-1 polypeptide and nesfatin-1M30 of human, mouse or rat, andtherefore antibodied obtained bind to all of these polypeptides. Whenadministered to an animal, this antibody exhibits an effect of enhancingfood intake and increasing body weight of said animal (Working Example14). In addition to them, it is possible to create antibody bypreparing, as appropriate, a peptide to be used as the antigen from thesequence of disclosed nesfatin-1 polypeptide etc.

A monoclonal antibody against the above nesfatin polypeptide etc. can beprepared by collecting immune cells from the animal sensitized with theantigen and subjecting the cells to cell fusion with myeloma cells tocollect the antibody from the culture.

Such an antibody can be labelled as appropriate, and can be used todetect the above nesfatin polypeptide etc. Also, instead of labellingthis antibody, substances that specifically bind to said antibody, suchas Protein A and protein G, can be labelled for indirect detection. Asspecific methods for detection, for example, ELISA methods can bementioned.

The antigen used to obtain the antibody of the present invention can beobtained by integrating, for example, a gene encoding the nesfatinpolypeptide, the nesfatin-1 polypeptide, nesfatin-1M30, nesfatin-1M16,nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16, NUCB1-M14 orNUCB1-M10M mentioned above, or altered products thereof, or part of itinto an expression vector, introducing the expression vector into asuitable host cell to construct a transformant, culturing saidtransformant to express a recombinant protein, and purifying theexpressed recombinant protein from the culture or the culturesupernatant. Alternatively, an oligopeptide can be chemicallysynthesized that comprises an amino acid sequence encoded by said geneor a partial amino acid sequence of the amino acid sequence encoded bythe full-length cDNA and used as the immunogen. Animals to be immunizedinclude mice, rats, rabbits, goats, horses, hamsters and the like, andcan be selected as appropriate by a person skilled in the art.

<A Substance that Suppresses the Activity or Expression of a PolypeptideHaving an Activity of Suppressing Food Intake and/or Suppressing BodyWeight Control>

In the investigation of the present invention, when an antibody wasprepared against a nesfatin polypeptide, a nesfatin-1 polypeptide and anesfatin-1M30 polypeptide, and administered to the brain of an animal,an increase in the amount of food intake by the animal was noted(Working Examples 7 and 14). Also, when an antisense RNA(oligonucleotide) that suppresses the expression of nesfatin wasadministered into the brain of an animal, the enhancement of food intakeand the increase in the body weight of the animal were noted (WorkingExample 15). This indicates that the nesfatin polypeptide, thenesfatin-1 polypeptide and the nesfatin-1M30 polypeptide actuallyoperates in the brain for food intake control and/or body weightcontrol. Furthermore, the increased concentration of the nesfatinpolypeptide, the nesfatin-1 polypeptide or the nesfatin-1M30 polypeptidein a peripheral (peripheral blood) or a brain caused the suppression offood intake and/or the suppression of body weight gain, and a substancethat suppresses the activity or expression of said polypeptide exhibitedan activity of enhancing food intake and/or increasing body weight, andtherefore the nesfatin polypeptide, the nesfatin-1 polypeptide and/orthe nesfatin-1M30 polypeptide was demonstrated to be a central factorresponsible for food intake control and/or body weight control.Accordingly, a substance per se that inhibits the activity or expressionof the nesfatin polypeptide, the nesfatin-1 polypeptide and/or thenesfatin-1M30 polypeptide can be used in the treatment, diagnosis and/orscreening of therapeutic agents for the suppression of food intakeand/or the suppression of body weight gain due to diseases andconditions for which decreased food intake and body weight reduction area problem, such as cibophobia, functional dyspepsia, cancer,inflammatory diseases, decreased functions of the pituitary, thethyroid, the adrenal gland etc., post-surgery, or excessive stress andthe like.

Thus, the present invention relates to a substance that inhibits theactivity or the production of the nesfatin polypeptide, the nesfatin-1polypeptide, the nesfatin-1M30 polypeptide, nesfatin-1M16,nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16, NUCB1-M14 orNUCB1-M10M. More preferably, it relates to a substance that exhibits anactivity of enhancing food intake and/or enhancing body weight gain.Substances that suppress the activity of the nesfatin polypeptide etc.include those characterized by binding to the nesfatin polypeptide etc.and those that do not require binding to these polypeptides. An exampleof the former is antibody against the nesfatin polypeptide etc., and asthe antibody those described above can be used. An example of the latteris a dominant negative polypeptide against the nesfatin polypeptide etc.The “dominant negative” is a mutant that dominantly acts on the wildtype both quantitatively and qualitatively so as to inhibit the functionof the wild type. The dominant negative can be prepared by deleting orconverting part of the amino acids of the wild type.

The present invention also relates to a substance that suppresses theexpression of a gene encoding the nesfatin polypeptide, the nesfatin-1polypeptide, the nesfatin-1M30 polypeptide, nesfatin-1M16,nesfatin-1M14, nesfatin-1M10M, NUCB1-M30, NUCB1-M16, NUCB1-M14 orNUCB1-M10M. Such substances include, for example, an antisenseoligonucleotide, a RNAi molecule and the like. The gene encoding thenesfatin polypeptide etc. as used herein refers to a nucleic acidmolecule that comprises the entire nucleotide sequence or part thereofof the nucleic acid molecule encoding the nesfatin polypeptide etc. as acontiguous or non-contiguous unit. Also the expression of gene comprisesa step of a nucleic acid molecule encoding the nesfatin polypeptide etc.being transcribed from said gene, a step of stabilizing the transcribednucleic acid molecule, and a step of the nesfatin polypeptide etc. beingproduced by translation from the transcribed nucleic acid molecule.Thus, the suppression of gene expression refers to suppress any of thesteps of transcription from the gene encoding the nesfatin polypeptideetc.; stabilization; and translation.

The above antisense oligonucleotide can be designed by using, forexample, a gene sequence encoding the nesfatin polypeptide. As such anantisense oligonucleotide, there can be mentioned a morpholino-typeantisense oligonucleotide having the structure of SEQ ID NO: 31. Asshown in Working Example 15, by administering this to an animal, saidantisense oligonucleotide exhibits an effect of enhancing food intakeand enhancing body weight gain. For an antisense oligonucleotides,various modifications or binding formats are known in order to avoiddecomposition in the cell, and a person skilled in the art will be ableto select the structure of a suitable antisense oligonucleotide (Currecket al., European Journal of Biochemistry) (UK), 2003, Vol. 270, pp.1628-486). As the structure, there can be illustrated the natural form(D-oligo), the phosphorothioate type (S-oligo), the methylphosphonatetype (M-oligo), the phosphoroamidate type (A-oligo), the 2′-O-methyltype (D-oligo), the morpholidate type (Mo-oligo), a polyamide nucleicacid and the like. The length used is 10 bases to 70 bases, andpreferably 15 bases to 30 bases are used.

RNA interference (RNAi) refers to a phenomenon in which adouble-stranded RNA of 21-23 residues decomposes a target RNA containingthe same sequence thereby to greatly suppress its expression. Thus, RNAcontaining a double stranded structure having the same nucleotidesequence as mRNA of the gene encoding the nesfatin polypeptide etc. canbe used for suppressing the expression of the gene of the nesfatinpolypeptide etc. In order to obtain the RNAi effect, it is preferred touse a double stranded RNA having a sequence of at least 20 contiguousnucleotides. The double stranded structure may be composed of differentstrands, and of two strands provided by the stem loop structure of oneRNA. By adding a two-base overhang to the 3′-end of each strand, theeffect of suppressing the expression of the gene can be enhanced. Forthe sequence, length and the structure used in designing RNAi, a personskilled in the art would be able to optimize RNAi having a potent effectof suppressing gene expression through various modification attempts.

The above antisense oligonucleotide molecule and the RNAi molecule canbe produced by integrating a nucleic acid molecule comprising anucleotide sequence complementary to the nucleic acid sequence of saidmolecule into a vector, introducing this into a host cell fortransformation, and culturing the transformant. The host cell used andthe method of transformation could be selected as appropriate by aperson skilled in the art as described in the above <Transformant>.

<A Pharmaceutical Product for Enhancing Appetite or Enhancing BodyWeight Gain>

The present invention also relates to a pharmaceutical composition forenhancing appetite or enhancing body weight gain, said compositioncomprising as an active ingredient a substance that inhibits theactivity or the production of the nesfatin polypeptide etc., or asubstance that inhibits the expression of the gene encoding the nesfatinpolypeptide etc. Said pharmaceutical composition can be used fordiseases and conditions for which the suppression of food intake or bodyweight gain is a problem. Diseases and conditions for which thesuppression of food intake or body weight gain is a problem include, forexample, cibophobia, functional dyspepsia, or the suppression of foodintake and/or the suppression of body weight gain due to cancer,inflammatory diseases, decreased functions of the pituitary, thethyroid, the adrenal etc., post-surgery, or excessive stress and thelike.

The pharmaceutical composition of the present invention may contain anypharmaceutically acceptable additives. Pharmaceutical formulations usingpharmaceutically acceptable additives may be prepared by a methoddescribed in “Remington: The Science and Practice of Pharmacy, 20thedition, University of the Sciences in Philadelphia, Williams & Wilkins,issued on Dec. 15, 2000”. One dosage form of such a pharmaceuticalcomposition is presented as a liquid prepared by dissolving, suspendingor emulsifying in an aqueous or oleaginous solution. Such solvents usedinclude, for example, distilled water for injection, physiologicalsaline etc. as an aqueous liquid, and in addition, an osmoregulatoryagent (for example, D-glucose, D-sorbitol, D-mannitol, and sodiumchloride), a suitable solubilizing agent such as alcohols (for example,ethanol), polyalcohols (for example, propylene glycol and polyethyleneglycol), nonionic surfactants (for example, polysorbate 80,polyoxyethylenated hydrogenated castor oil 50) and the like may be used.Also, as the solvents, an oleaginous solution may be used, and saidoleaginous solution includes sesame oil, soybean oil etc., and as thesolubilizing agent, benzyl benzoate, benzyl alcohol etc. may be used incombination. In such liquids, there can be used, as appropriate,additives such as buffers (such as phosphate buffers and acetatebuffers), soothing agents (such as benzalkonium chloride and procainehydrochloride), stabilizers (such as human serum albumin andpolyethylene glycol), preservatives (such as ascorbic acid, erythorbicacid, and salts thereof), colorants (such as copper chlorophyll,β-carotene, Red No. 2, and Blue No. 1), preservatives (such as ascorbicacid, erythorbic acid, and salts thereof), antiseptics (such asparaoxybenzoate ester, phenol, benzethonium chloride and benzalkoniumchloride), thickeners (such as hydroxypropyl cellulose, carboxymethylcellulose, and salts thereof), stabilizers (such as human serum albumin,mannitol and sorbitol), and corrigents (such as menthol and citrusflagrances). Another dosage form of pharmaceutical compositions (inorder to unify the expression with the above several “pharmaceuticalcompositions”) includes solid forms such as powders, tablets, granules,capsules, pills, suppositories, and lozenges. In the case of solid formsfor administration in the form of oral preparations, additives usedinclude excipients (such as crystalline cellulose, lactose and starch),lubricants (such as magnesium stearate and talc), binders (such ashydroxypropyl cellulose, hydroxypropyl methyl cellulose and macrogol),disintegrants (such as starch and carboxymethyl cellulose calcium), andthe like. Also, as needed, there can be used antiseptics (such as benzylalcohol, chlorobutanol, methyl paraoxybenzoate and propylparaoxybenzoate), antioxidants, colorants, sweeteners and the like.Furthermore, another form also includes a pharmaceutical composition forapplication to the mucosa, and for this form of formulations, in orderto impart adsoptivity to the mucosa and retentivity, there can becontained additives such as tackifiers, tackiness enhancers, thickeners,thickening agents (such as mucin, agar, gelatin, pectin, caragennan,sodium alginate, locust bean gum, xanthan gum, tragacanth gum, gumarabic, chitosan, pullulan, waxy starch, sucralfate, cellulose and itsderivatives (such as hydroxypropyl methyl cellulose), polyglycerin fattyacid esters, acrylic-(meth)acrylic alkyl copolymers or salts thereof,and polyglycerin fatty acid esters). However, dosage forms ofpharmaceutical compositions and solvents and additives to be deliveredto the living body are not limited to the above, and can be selected, asappropriate, by a person skilled in the art.

For the purpose of ameliorating disease conditions, the abovepharmaceutical compositions can be administered orally or parenterally.In the case of oral administration, dosage forms such as granules,powders, tablets, capsules, liquids, syrups, emulsions or suspensions,and elixirs can be selected. In the case of parenteral administration,it can be transnasal agents, and liquids, suspensions or solidformulations can be selected. Other agents in the form of parenteraladministration can be in the form of injections, and injections selectedmay be hypodermic injections, intravenous injections, drip injections,intramuscular injections, intraventricular injections, orintraperitoneal injections and the like. Other formulations for use inparenteral administration include, for example, transmucosalpreparations other than suppositories, sublingual tablets, transdermalpreparations, transnasal preparations and the like. Furthermore,intravascular local administration can be performed in embodimentswherein they are contained or applied to stents, or anti-intravascularembolism agents.

The dosage of the above pharmaceutical composition may vary depending onthe age, sex, body weight of the patient, condition, therapeutic effect,administration regimen, treatment period, or the types of activeingredients contained in said pharmaceutical composition, but its onedosage is usually in the range of 0.1-500 mg per person for adults, andpreferably in the range of 0.5 mg-20 mg. The dosage may vary dependingon various conditions, and thus a dosage smaller than that describedabove may sometimes be sufficient, or at other times a dosage greaterthan the above may be required.

The above antisense oligonucleotide or the RNAi molecule may beintegrated downstream to a suitable promoter sequence, and can beadministered as the RNA expression vector that provides the effect ofantisense oligonucleotide or RNAi. When the expression vector isintroduced in a form that can reach the brain of the subject patient,the effect of the antisense oligonucleotide or RNAi effect of the geneallows the expression of a polynucleotide that suppresses the expressionof said gene, and the reduced expression level of said gene can attainthe therapeutic effect for conditions of suppressed food intake and/orsuppressed body weight gain.

<Transgenic Animals, Animal Model of Obese/Adiposis>

The present invention relates to a transgenic non-human animalcomprising a gene encoding the above nesfatin polypeptide etc., or avector containing them. More specifically, it relates to a transgenicnon-human animal that exhibits the state of suppressed food intake orthe state of suppressed body weight gain wherein the expression of saidgene is enhanced systemically, preferably, in the hypothalamus. Atransgenic non-human animal in which the expression level of the geneencoding the above polypeptide has been artificially enhanced can beused as an animal model that exhibits suppressed food intake and/orsuppressed body weight gain.

The present invention relates to a transgenic non-human animal that hasintroduced therein the above substance (such as antibody, antisenseoligonucleotide, and RNAi molecule) that suppresses the activity or theexpression of a polypeptide having an activity of suppressing foodintake and/or suppressing body weight control, and that exhibitsenhanced appetite or enhanced body weight gain. A non-human transgenicanimal produced by introducing a gene encoding such a substance can beused as an animal model that exhibits enhanced food intake or enhancedbody weight gain. Also, said transgenic non-human animal can be used asan animal model for diseases such as obesity, diabetes mellitus,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases such as osteoarthritis, menstrual disorders and malignanttumors.

The method of obtaining transgenic animals with a specific gene as thetarget is known. Thus, transgenic animals can be obtained by a method inwhich a gene and an egg are mixed and then treated with calciumphosphate, a method in which a gene is directly introduced into thenucleus of the egg in the pronucleus phase with a micropipette under aphase contrast microscope (the microinjection method, U.S. Pat. No.4,873,191), a method of using an embryonic stem cell (ES cell), and thelike. In addition, there have been developed a method in which a gene isinserted into a retrovirus vector and then infected to the egg, asperm-vector method in which a gene is introduced into the egg via thesperm, and the like. The sperm-vector method is a gene recombinantmethod in which a foreign gene is incorporated into the sperm byadhesion or electroporation and then the sperm is allowed to fertilizethe egg to introduce the foreign gene (Lavitranoet M. et al., Cell(1989) 57, 717-723).

Also, if a promoter of which transcription is regulated by a suitablesubstance such as a drug is used as a promoter for use in the expressionvector, the administration of said substance can regulate the expressionlevel of the substance that controls the activity or expression of thegene encoding a foreign nesfatin polypeptide etc. or said polypeptide ina transgenic animal.

Furthermore, the present invention relates to a knock-out animal whereinthe entire region or part thereof of the gene encoding the nesfatinpolypeptide etc. has been deleted, and a knock-in animal in which saidgene has been replaced with another gene is also encompassed in thepresent invention. For example, a knock-out animal of the nesfatinpolypeptide etc. can be used as an animal model that exhibits enhancedfood intake and/or enhanced body weight gain.

Furthermore, the present invention also relates to an animal model inwhich food intake and/or body weight gain has been suppressed comprisinga non-human animal in which the above nesfatin polypeptide etc. per sehas been administered, or an animal model in which food intake and/orbody weight gain has been enhanced comprising a non-human animal inwhich a substance that suppresses the activity or expression of saidpolypeptide has been administered. The non-human animal in which foodintake and/or body weight gain has been enhanced can be used as ananimal model for diseases such as obesity, diabetes mellitus,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases such as osteoarthritis, menstrual disorders and malignanttumors.

The animal species of the present invention for use as an animal modelcan be prepared using any vertebrate other than a human. Specifically,for vertebrates such as mice, rats, rabbits, minipigs, goats, sheep,monkeys, dogs, cats, and cattle, animal models can be prepared by theintroduction of a gene or the administration of a substance.

<Method of Producing a Polypeptide Having an Activity of SuppressingFood Intake and/or Suppressing Body Weight Gain>

The present invention relates to a method of producing the abovenesfatin polypeptide etc., using the above transformant that expressesthe gene encoding said nesfatin polypeptide etc., or the abovetransgenic non-human animal and a transgenic plant comprising the geneencoding said polypeptide or a vector containing the gene.

In the above method of producing said polypeptides of the presentinvention, various modifications and alterations can be made to theabove DNA sequence, the plasmid and the virus in order to be compatiblewith expression, transcription, translation etc. in the transformant orthe transgenic non-human animal. For example, due to degeneracy of thegenetic code, nucleotides can be substituted through the entire codingregion of protein. Such a sequence can be predicted from the amino acidsequence of the nesfatin polypeptide or nesfatin-1 polypeptide, or fromthe nucleotide sequence of the gene encoding said polypeptide, and canbe assembled by a conventional synthetic method described below. Such asynthetic method can be performed by the method of Itakura et al.(Itakura et al., Science 198:1059, 1977) and the method of Crea et al.(Crea et al., Proc. Natl. Acad. Sci. USA 75:5765, 1978). Thus, the geneencoding the above nesfatin polypeptide etc. for use in the method ofproducing the polypeptide of the present invention is not limited tothose employing the specifically illustrated nucleotide sequence, theplasmid or the virus.

The production of the above polypeptide using a transformant can beperformed by culturing said transformant. As the culture medium used forculturing, various media commonly used may be selected as appropriatedepending on the host cell adopted, and the culturing may be performedunder a condition suitable for the growth of the host cell.

Also, the above polypeptide can be produced in the transformantintracellarly or extracellary, or on the cell membrane. As other methodsof producing polypeptides using a gene encoding the nesfatin polypeptideetc., there can be mentioned a method based on cell-free proteinsynthesis, a representative of which is an in vitro translation reactionsystem. In this in vitro translation reaction system, 5′ upstream to thegene encoding the nesfatin polypeptide etc., a sequence that controlstranscription, preferably SP6 promoter, T3 promoter, T7 promoter etc.may be added, and the gene is transcribed into the cell or in vitro toprepare a RNA molecule encoding the nesfatin polypeptide etc., and acell extract for in vitro transcription prepared from wheat germ, E.coli, reticulocytes etc. to carry out the production. One example of theproduction can be accomplished by the method described in Sawazaki etal., Protein, Nucleic acid and Enzyme, 2003, Vol. 48, pp. 549-554.Polypeptides produced by such a transformant or cell-free proteinsynthesis can be separated and purified as desired by various separationprocesses using the physical properties, chemical properties etc. [seeThe Japanese Biochemical Society ed., “Biochemistry Databook II”, TheFirst edition, the First print, issued by Tokyo Kagaku Dojin Co., Ltd.on Jun. 23, 1980, pp. 1175-1259; Arakawa et al., Biochemistry (USA),issued on Dec. 16, 1986, Vol. 25, No. 25, pp. 8274-8277 (1986); Langleyet al., European Journal of Biochemistry (Germany), issued on Mar. 2,1987, Vol. 163, No. 2, pp. 313-321]. Said methods specifically include,for example, conventional reconstitution treatment, treatment with aprotein precipitating agent (salting out), centrifugation, the osmoticshock method, ultrasonic disruption, ultrafiltration, gel filtration,various chromatographic methods such as adsorption chromatography, ionexchange chromatography, affinity chromatography, and high performanceliquid chromatography (HPLC), dialysis, and combinations thereof, andthe like. Purification using affinity with the above polypeptide canuse, for example, the above-mentioned antibody that binds to the abovepolypeptide, and can be accomplished by desorption of said polypeptideand said antibody.

The above method of producing the polypeptide of the present inventioncan also be accomplished by producing a protein in which an affinity tagwas fused to said polypeptide in a transformant or a transgenicnon-human animal, and then separating and purifying said affinitytag-fused protein. By expressing said affinity tag-fused protein,affinity purification using this tag can be performed. As said affinitytag, there can be mentioned glutathione S-transferase (GST),poly-Histidine (His tag, Sisk et al., J. Virol. (USA), issued inFebruary 1994, Vol. 68, No. 2, pp. 766-775) and FLAG tag (Hopp et al.,Biotechnology, issued in 1988, Vol. 6, pp. 1204-1210).

In the case of a GST-fused protein, in which the above polypeptide wasfused to GST, the above polypeptide can be produced using aglutathione-immobilized carrier, i.e. said polypeptide can be purifiedusing absorption and desorption of GST and the glutathione boundcarrier, and then cleaving the GST portion from the GST-fused proteinand purifying it.

In the case of a His tag-fused protein, the above polypeptide can beproduced using a metal ion chelate carrier, i.e. said polypeptide can bepurified using absorption and desorption of the His tag and said metalion chelate carrier, and then cleaving the His tag portion from the Histag-fused protein and purifying it.

In the case of a FLAG tag-fused protein, the above polypeptide can beproduced using a carrier to which anti-FLAG tag antibody is bound, i.e.said polypeptide can be purified using absorption and desorption of theFLAG tag and said metal ion chelate carrier, and then cleaving the FLAGtag portion from the FLAG tag-fused protein and purifying it.

The nesfatin-1 polypeptide can also be prepared by treating the nesfatinpolypeptide obtained in the above-mentioned method with a proteolyticenzyme such as protein convertase, fractionating the digest by a reversephase chromatography, and confirming and collecting fragments containingthe nesfatin-1 polypeptide by mass spectrometry etc. The alterednesfatin-1 can also be obtained in a similar manner.

The thus obtained nesfatin polypeptide etc. of the present invention canalso be modified at the N-terminal after translation, and such modifiedpolypeptide molecules are also encompassed in the present invention. Forexample, a polypeptide of which N-terminal has been converted topyroglutamine can be obtained by expressing the above nesfatinpolypeptide etc. of the present invention etc. so that the N-terminalbecomes a glutamine residue, and treating the polypeptide obtained underan acidic condition of a 5-10% acetic acid solution (Park et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica (USA), issued in March 1991, pp. 2046-2050). A peptide of whichN-terminal has been acetylated can be obtained by expressing the abovenesfatin polypeptide etc. of the present invention so that theN-terminal becomes any amino acid having an α-amino group, and treatingthe polypeptide obtained with sulfo-NHS-acetate or acetic anhydride.Such a method of modifying the N-terminal of the polypeptide aftertranslation is well known in the art. In addition, the nesfatinpolypeptide etc. of the present invention can also be treated with afluorescent substance to be modified (Hermanson et al., BioconjugateTechniques (USA) issued by Academic Press in 1996).

A transgenic non-human animal can also be used to produce the abovenesfatin polypeptide etc. According to such a method, for example,fractions containing the peptide of interest can be collected as aliquid component separated from the solid component, or as a liquidcomponent extracted with an aqueous solvent or an organic solvent fromprocessed products obtained by cutting, milling, or fractionating anorgan, tissue, blood, milk etc. harvested from a transgenic non-humananimal having a gene encoding the above nesfatin polypeptide etc. or avector comprising said gene. The peptide of interest can be separatedand purified from said fractions by various separation procedures usingthe above-mentioned physical properties, chemical properties etc.

It is also possible to use a transgenic plant to produce the abovenesfatin polypeptide etc. The method can be performed referring toJapanese Unexamined Patent Publication (Kokai) No. 2003-116385 “Atransgenic plant comprising a gene encoding Japanese encephalitisvaccine”. Also, a transgenic plant can produced by referring to JapaneseUnexamined Patent Publication (Kokai) No. 2002-17186 “TransgenicPlants”. From the tissue of leaves, stems, roots, fruits, rinds, sproutsand petals of such a transgenic plant or callus tissue derived from sucha tissue, or optionally from processed products obtained by cutting,peeling, milling, or compressing of said tissue, the polypeptide ofinterest can be collected in an extract with an aqueous solvent or anorganic solvent, a squeeze obtained by the compression or an oil, andfurthermore said polypeptide can be separated and purified by variousseparation procedures using the above-mentioned physical properties,chemical properties etc.

The above polypeptide of the present invention can also be obtained bychemical synthesis. In this case, a commonly used peptide syntheticmethod such as solid-phase synthesis or liquid-phase synthesis can beused. For the condensation and protection of amino acid protectinggroups in peptide synthesis and the elimination of the protecting groupsafter synthesis, a known method can be used (Izumitani et al., “Basisand Experiment of Peptide Synthesis (PEPUTIDO GOSEINO KISOTO JIKKENN)”,issued by Maruzen Co., Ltd. in 1975, Hitoshi Yajima—The JapaneseBiochemical Society ed., “Biochemistry Experiment Series (SEIKAGAKUJIKKENN KOUZA) 1, Chemistry of Protein IV”, issued by Tokyo Kagaku DojinCo., Ltd. in 1977). It is also possible to synthesize the entire peptidesequence at one time, while a method can be used in which partialpeptides of said protein are synthesized separately and then the partialpeptides are condensed (The Japanese Biochemical Society ed., “NewBiochemistry Experiment Series (SINSEIKAGAKU JIKKENN KOUZA), Protein IV,Synthesis and Expression”, issued by Tokyo Kagaku Dojin Co., Ltd. in1991).

Though the α-amine of amino acids commonly used in peptide synthesis isusually protected with a tBoc group or a Fmoc group, the peptide to befinally obtained may retain the protecting group or may be deprotected.As needed, the deprotected amino terminal of the peptide may be modifiedwith pyroglutamic acid, an acetyl group or a formyl group (Hermanson etal., Bioconjugate Techniques (USA) issued by Academic Press in 1996). Ina specific example, the above polypeptide of the present inventionhaving glutamine at the N-terminal is synthesized, and said peptide canbe cyclized by treating with a dilute acid such as a 5-10% acetic acidto convert to pyroglutamic acid (Park et al., Proceedings of theNational Academy of Sciences of the United States of America (USA),issued in March 1991, pp. 2046-2050).

Also, as needed, synthetic peptides in which an amide group or afluorescent substance has been added at the C-terminal of thepolypeptide after synthesis can be prepared. For example, in order toobtain a synthetic peptide having introduced therein an amide group atthe C-terminal by solid-phase synthesis, a commercial resin that allowsthe amidation of the C-terminal can be used in a reaction of cleavingthe peptide from the solid-phase carrier (resin). Also, modificationwith fluorescent substances etc. at the C-terminal can be carried out bya known method (Hermanson et al., Bioconjugate Techniques (USA) issuedby Academic Press in 1996).

<Assay Method of Predicting or Diagnosing the State of Food IntakeControl and/or Body Weight Control>

The present invention relates to a diagnostic method for use in thejudgment of diseases associated with food intake control and/or bodyweight control in mammals, the monitoring and prediction of severity andprogress of disease conditions, the judgment of prognosis and theadministration of the above pharmaceutical composition and the like.Thus, the present invention relates to a method of predicting ordiagnosing the state of food intake control and/or body weight controlusing a sample derived from a mammal to be tested, which methodcomprises comparing the amount contained of a nucleic acid molecule (anucleic acid molecule comprising a nucleotide sequence set forth in anyof SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124) encodingnesfatin, the nesfatin polypeptide etc. (a polypeptide comprising anamino acid sequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15,39-41, 65-73, 101-103 and 107-115) in said sample with that in a samplederived from a normal individual. A normal individual as used hereinrefers to a normal or untreated individual.

Specifically, the present invention relates to an assay method ofpredicting or diagnosing the state of food intake control and/or bodyweight control, which method comprises the steps of comparing the amountcontained of a nucleic acid molecule comprising a nucleotide sequenceset forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and116-124 in a sample derived from a living body of a mammal to be testedwith that in a biological sample derived from a normal individual, anddetecting the decreased or the enhanced expression of said gene.

As biological materials to be used, there can be mentioned blood, urine,cerebrospinal fluid, saliva, brain tissues harvested from biopsy etc.with blood being most preferred. Blood for use as a sample includeswhole blood, or plasma or serum derived from whole blood. Methods ofcollecting these biological samples are known. Preparations such aslysates of these biological samples can also be used as the sample.Alternatively, mRNA extracted from the preparations may be used as asample for determining mRNA corresponding to the above gene. For theextraction of lysates of biological samples or mRNA, commerciallyavailable kits can be conveniently used. Alternatively, liquidbiological samples such as blood and cerebrospinal fluid can be dilutedas needed with a buffer etc. to prepare samples for determining proteinor gene.

The amount contained of a nucleic acid molecule comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 and 116-124 in a biological sample of a mammal can be determinedby using a polynucleotide comprising the nucleotide sequence of saidnucleic acid molecule or an oligonucleotide with a length of at least 18bases comprising a nucleotide sequence complementary to thecomplementary chain as a PCR primer or a probe. In this case, a skilledin the art can design a primer or a probe suitable for an application,using variety of computer program. Such a polynucleotide or anoligonucleotide may be bound to a suitable label or immobilized to asuitable support depending on the assay format. Such a PCR primer or aprobe can be recombinantly or synthetically produced, or may be producedby any means available to a person skilled in the art. For example, thePCR primer that can be used in the present invention include, but notlimited to, SEQ ID NO: 22 (forward primer) and SEQ ID NO: 23 (reverseprimer). As the probe that can be used in the present invention include,but not limited to, one that is obtained by labelling a fragmentamplified using the above primer with the DNA sequence of SEQ ID NO: 21as the template (see Working Example 2).

The measured values of expression level of a nucleic acid moleculecomprising a nucleotide sequence set forth in any of SEQ ID NOs: 10-12,18-20, 44-46, 74-82, 104-106 and 116-124 can be corrected using a knownmethod. By performing correction, the expression level of a nucleic acidmolecule comprising a nucleotide sequence set forth in any of SEQ IDNOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 can be comparedbetween the biological sample to be tested and the biological samplefrom a normal individual. The correction of the measured values may becarried out based on the measured value of the expression level of agene (such as the housekeeping gene) of which expression level does notgreatly vary. Examples of the gene of which expression level does notgreatly vary include, for example, β-actin and GAPDH.

When mRNA is used in the determination of expression level of a nucleicacid molecule comprising a nucleotide sequence set forth in any of SEQID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124, a formatcompatible with the method of diagnosis may be carried out asappropriate. When, for example, mRNA extracted from the biologicaltissue is used, the RT-PCR method or the Northern blotting method etc.can be selected, and when tissue specimens are used, the in situhybridization method etc. can be selected. For example, Working Example8 discloses that in situ hybridization method indicates that anexpression of the nesfatin gene is suppressed in rats enhanced appetiteby fasting, and the expression is enhanced in a state of suppressedappetite by food intake.

When the expression of a gene comprising a nucleotide sequence set forthin any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 and 116-124 ina test sample was detected to be decreased compared to that of abiological sample derived from a normal individual, the state ofenhanced food intake or enhanced body weight gain is predicted ordiagnosed, and furthermore the onset of a disease selected from obesity,diabetes mellitus, hypertension, hyperlipidemia, hyperuricemia, fattyliver, cardiac diseases, cerebral vascular diseases, sleep apneasyndrome, orthopedic diseases, menstrual disorders and malignant tumorscan be predicted or diagnosed. On the other hand, when the expression ofsaid gene in a biological sample was found to be increased, the mammaltested is in the state of suppressed food intake or suppressed bodyweight gain is predicted or diagnosed, and furthermore, cibophobia,functional dyspepsia, or the disease conditions of suppressed foodintake or suppressed body weight gain resulting from cancer,inflammatory diseases, decreased functions of the pituitary, thethyroid, the adrenal etc., post-surgery, or excessive stress and thelike can be predicted or diagnosed.

Furthermore, the present invention relates to a method of predicting ordiagnosing the state of food intake control and/or body weight control,which method comprises a step of detecting the decreased state or theenhanced state of the amount contained of the nesfatin polypeptide etc.(a polypeptide comprising an amino acid sequence set forth in SEQ IDNOs: 3, 6, 9, 13-15, 39-41, 65-73, 101-103 or 107-115) by comparing theamount contained of said polypeptide in biological sample of a mammalwith that in a biological sample derived from a normal individual. Saidmethod can be carried out by determining the nesfatin polypeptide etc.

As biological samples to be used, there can be mentioned blood, urine,cerebrospinal fluid, saliva, brain tissues harvested from biopsy etc.with blood being most preferred. Blood for use as a sample includeswhole blood, or plasma or serum derived from whole blood. Methods ofcollecting these biological samples are known. Preparations such aslysates of these biological samples can also be used as the sample. Forthe preparation of lysates of biological samples, commercially availablekits can be conveniently used. Alternatively, liquid biological samplessuch as blood and cerebrospinal fluid can be diluted as needed with abuffer etc. to prepare samples for determining protein or gene.

In order to determine the nesfatin polypeptide etc., an immunologicalmethod for said polypeptide can be used in which an antibody that bindsto the nesfatin polypeptide etc. can be used. Such an antibody includesthose mentioned above, and specifically there can be mentioned anantibody obtained by immunizing a peptide derived from the nesfatinpolypeptide obtained in Working Examples 3 and 10, or an antibodyobtained by immunizing a peptide derived from the nesfatin-1 polypeptideor the nesfatin-1M30 polypeptide. These antibodies are not limiting, andany polyclonal antibody or monoclonal antibody against the nesfatinpolypeptide etc. can be used without specific limitation.

The antibody for use in the present invention may be bound to a suitablelabel depending on the assay format, or said antibody may be immobilizedto a suitable support depending on the assay format.

An immunological method can be carried out as appropriate in a formatcompatible with the method of diagnosis. For example, when blood samplesor lysates are used, the ELISA method, the RIA method, the Westernblotting method etc. can be selected, and when tissue specimens areused, immunohistochemical methods etc. may be selected. In a morespecific example, the Western blotting method as shown in Examples, theimmunohistochemical method as shown in Working Examples 4 and 11 can bementioned, and it is possible to construct an assay system for thenesfatin polypeptide etc. using appropriate using antibody. For example,the Western blotting method is shown in Working Example 3,immunohistochemical methods in Working Examples 4 and 9, and the ELISAmethod (competitive EIA method) in Working Example 21.

When the amount contained of the nesfatin polypeptide or the nesfatinpolypeptide etc. in a biological sample was found to be decreasedcompared to that of a biological sample derived from a normalindividual, the state of enhanced food intake or enhanced body weightgain is predicted or diagnosed, and furthermore the onset of a diseaseselected from obesity, diabetes mellitus, hypertension, hyperlipidemia,hyperuricemia, fatty liver, cardiac diseases, cerebral vasculardiseases, sleep apnea syndrome, orthopedic diseases, menstrual disordersand malignant tumors is predicted or diagnosed. On the other hand, whenthe amount contained of said polypeptide in a biological sample wasfound to be increased, the state of suppressed food intake or suppressedbody weight gain is predicted or diagnosed, and furthermore, cibophobia,functional dyspepsia, or the disease conditions of suppressed foodintake or suppressed body weight gain resulting from cancer,inflammatory diseases, decreased functions of the pituitary, thethyroid, the adrenal etc., post-surgery, or excessive stress and thelike can be predicted or diagnosed.

<Kit for Use in the Method of Predicting or Diagnosing the State of FoodIntake Control and/or Body Weight Control>

The present invention also relates to a kit for use in the method ofpredicting or diagnosing the above state of food intake control and/orbody weight control. Such a kit includes, for example, a kit fordetecting the amount expressed of the gene encoding nesfatin etc. (agene comprising a nucleotide sequence set forth in any of SEQ ID NOs:10-12, 18-20, 44-46, 74-82, 104-106 and 116-124) or a kit for detectingthe amount contained of the nesfatin polypeptide etc. (a polypeptidecomprising an amino acid sequences set forth in any of SEQ ID NOs: 3, 6,9, 13-15, 39-41, 65-73, 101-103 and 107-115).

The kit for detecting the amount expressed of the gene encoding thenesfatin etc. includes at least one of a PCR primer, a probe or a DNAchip for detecting said gene.

The PCR primer contained in the kit of the present invention refers toan oligonucleotide with a length of at least 18 bases comprising thenucleotide sequence of a gene encoding nesfatin etc. or a nucleotidesequence complementary to the complementary chain thereof, and would beprepared as appropriate based on the base sequence of a gene encodingnesfatin etc. described herein by a person skilled in the art. Forexample, the PCR primers contained in the kit of the present inventioncan include, but are not limited to, SEQ ID NO: 22 (forward primer) andSEQ ID NO: 23 (reverse primer). The probe contained in the kit of thepresent invention includes, but not limited to, one that is obtained bylabelling a fragment amplified using the above primer and the DNAsequence of SEQ ID NO: 21 as the template. The DNA chip contained in thekit of the present invention can be prepared by immobilizing the aboveprobe onto a substrate such as glass.

In addition, the kit of the present invention may include, as additionalelements, a buffer for diluting reagents and biological samples,positive controls, negative controls, a substrate for determining thelabel, a reaction vessel, an instruction describing the assay protocoletc. These elements may be mixed in advance as needed. Also, as needed,preservatives and antiseptics may be added to each element.

The kit for determining the amount produced of the nesfatin polypeptideetc. includes an antibody that recognizes said polypeptide, a standardpeptide and at least one modified peptide for competitive bindingreaction.

The antibody contained in the kit of the present invention, similarly tothe antibody used the above method, may be, but is not limited to, anantibody that recognizes the nesfatin polypeptide etc.

The standard peptide contained in the kit of the present invention isused for constructing a calibration curve that demonstrates a dosedependent binding of the above antibody to the nesfatin polypeptide etc.Such a standard polypeptide includes, for example, a polypeptiderecognized by the above antibody such as the nesfatin polypeptide etc.

The modified peptide for competitive binding reaction, contained in thekit of the present invention, refers to a peptide recognized by theabove antibody, wherein the peptide has an ability of competing with thenesfatin polypeptide or the like in biological samples to be determinedfor binding to said antibody. Also, the modified peptide for competitivebinding reaction is usually used by labelling it as appropriate.

The kit of the present invention can determine the amount of thenesfatin polypeptide etc. contained in a biological sample by, forexample, (1) constructing a calibration curve obtained from the standardpeptide and the above antibody in the presence of the above modifiedpeptide for competitive binding reaction, (2) adding the above antibodyin the presence of the above modified peptide for competitive bindingreaction in the biological sample, and (3) determining the amount boundof the modified peptide for competitive binding reaction and theantibody using the standard calibration curve.

It is also possible to determine the amount of the nesfatin polypeptideetc. using two kinds of antibodies that recognize the nesfatinpolypeptide or the like and that do not inhibit each other. In thiscase, the primary antibody is usually immobilized on a solid-phase, andthe secondary antibody is suitably labelled for use.

The kit of the present invention can determine the amount of thenesfatin polypeptide etc. contained in a biological sample by, forexample, (1) constructing a standard calibration curve by adding thestandard peptide to the immobilized primary antibody and reacting thepeptide with the primer, and then adding a labelled secondary antibody,(2) adding and reacting the biological sample to the similarlyimmobilized primary antibody and then adding the labelled secondaryantibody, and (3) determining the amount bound of the secondary antibodyin the reaction with the biological sample using the calibration curve.

In addition, the kit of the present invention may include, as additionalelements, a buffer for diluting reagents and biological samples,positive controls, negative controls, a substrate for determining thelabel, a reaction vessel, an instruction describing the assay protocoletc. These elements may be mixed in advance as needed. Also, as needed,preservatives and antiseptics may be added to each element.

<Method of Screening a Candidate Therapeutic Compound>

The present invention relates to a method of screening a therapeutic orpreventive candidate compound for diseases or conditions associated withfood intake control and/or body weight control, said compound having aneffect of food intake control and/or body weight control.

Thus, the present invention relates to a method of screening atherapeutic or preventive agent having an effect of suppressing foodintake and/or suppressing body weight gain, said method comprising thesteps of contacting a test substance with a mammalian cell, anddetecting the induced expression of the gene (a gene comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 and 116-124) encoding nesfatin etc. in said cell or anincrease in the amount of the nesfatin peptide etc. (a polypeptidecomprising an amino acid sequence set forth in any of SEQ ID NOs: 3, 6,9, 13-15, 39-41, 65-73, 101-103 and 107-115) contained in said cell orextracellularly secreted out of the cell. It has been demonstrated thatthe suppression of food intake or the suppression of body weight gain isrequired in obesity, diabetes mellitus, hypertension, hyperlipidemia,hyperuricemia, fatty liver, cardiac diseases, cerebral vasculardiseases, sleep apnea syndrome, orthopedic diseases, menstrual disordersand malignant tumors. By the above screening method, a therapeutic orpreventive agent for said diseases can be obtained.

On the other hand, the present invention also relates to a method ofscreening a therapeutic or preventive agent having an effect ofenhancing food intake and/or enhancing body weight gain, said methodcomprising the steps of contacting a test substance with a mammaliancell, and detecting the suppressed expression of the gene encodingnesfatin etc. in said cell or a decrease in the amount of the nesfatinpeptide etc. contained in said cell or extracellularly secreted out ofthe cell. It has been demonstrated that the enhancement of food intakeor the enhancement of body weight gain is required in cibophobia,functional dyspepsia, or the state of suppressed food intake orsuppressed body weight gain resulting from cancer, inflammatorydiseases, decreased functions of the pituitary, the thyroid, the adrenaletc., post-surgery, or excessive stress and the like. By the abovescreening method, a therapeutic or preventive agent for said diseasescan be obtained.

Compounds that either increase or decrease the expression level of saidpolypeptide or a gene encoding it are compounds that act in a promotingor suppressive manner on any step of gene transcription, stabilizationor translation, and the secretion, activity expression or stabilizationof protein. As used herein, compounds that decrease the expression levelof a gene are compounds that have an inhibitory effect on any of thesesteps.

The method of screening a candidate therapeutic compound of the presentinvention for diseases or conditions associated with food intake controland/or body weight control may be carried out in vivo or in vitro. Thisscreening in an in vivo case, for example, may be carried out accordingto the following steps:

(1) a step of administering a candidate compound to a test animal;

(2) a step of determining the expression intensity of the nesfatinpolypeptide etc., or the gene encoding said polypeptide in a biologicalsample from the above test animal; and

(3) a step of selecting a compound that enhances, or a compound thatdecreases, the expression intensity of the nesfatin polypeptide etc., orthe gene encoding said polypeptide, as compared to the control thatreceived no candidate compound.

A compound that enhances the production of the nesfatin polypeptide etc.and the expression intensity of the gene encoding said polypeptide canbe a candidate of a therapeutic agent for suppressing food intake and/orsuppressing body weight gain. Substances having such an activityinclude, but not limited to, a PPARγ agonist and the like. On the otherhand, a compound that suppresses the production of the nesfatinpolypeptide etc. or decreases the expression intensity of the geneencoding said polypeptide can be a candidate of a therapeutic agent forenhancing food intake and/or enhancing body weight gain. Substanceshaving such an activity include, but not limited to, an antibody thatbinds to the nesfatin polypeptide etc., an antisense oligonucleotideagainst the gene encoding said polypeptide, a PPARγ antagonist and thelike.

As the test animal for use in the screening method of the presentinvention, normal animals can be used and pathological animal models fordiseases associated with food intake control and/or body weight controlcan also be used as appropriate. As the example of such pathologicalanimal models, there can be mentioned the C57BL/6L Ham Slc^(AY) obesemice, the Zucker-fa/fa obese rats and the like. Also the transgenicanimals of the present invention or animal models that received thenesfatin polypeptide etc., or a substance that suppresses the activityor the expression of said polypeptide can be used.

The determination of expression intensity of the nesfatin polypeptideetc., or the gene encoding said polypeptide can be carried out by animmunological method or by determining mRNA. In the immunologicalmethod, a format compatible with the purpose of determination or thebiological sample may be used as appropriate. For example, when bloodsamples or lysates of biological tissues are used as the biologicalsample, the ELISA method, the RIA method, the Western blotting methodetc. can be selected, and when tissue specimens are used,immunohistochemical methods etc. may be selected. When mRNA is to bedetermined, a format compatible with the purpose of determination or thebiological sample may be used as appropriate. When, for example, mRNAextracted from the biological tissue is used, the RT-PCR method or theNorthern blotting method etc. can be selected, and when tissue specimensare used, the in situ hybridization method etc. can be selected.

An example of in vitro screening can be carried out according to thefollowing steps:

(1) a step of contacting a candidate compound with a mammal-derived cellor tissue;

(2) a step of determining the expression intensity of the nesfatinpolypeptide etc., or the gene encoding said polypeptide in the abovecell sample or the tissue sample; and

(3) a step of selecting a compound that enhances, or a compound thatdecreases, the expression intensity of the nesfatin polypeptide etc., orthe gene encoding said polypeptide, as compared to the control thatreceived no candidate compound.

In this case, a compound that enhances the production of the nesfatinpolypeptide etc. and the expression intensity of the gene encoding saidpolypeptide can be a candidate of a therapeutic agent for suppressingfood intake and/or suppressing body weight gain. Substances having suchan activity include, but not limited to, a PPARγ agonist and the like.On the other hand, a compound that suppresses the production of thenesfatin polypeptide etc. or decreases the expression intensity of thegene encoding said polypeptide can be a candidate of a therapeutic agentfor enhancing food intake and/or enhancing body weight gain. Substanceshaving such an activity include, but not limited to, an antibody thatbinds to the nesfatin polypeptide etc., an antisense oligonucleotideagainst the gene encoding said polypeptide, a PPARγ antagonist and thelike.

As the test cell for use in the screening method of the presentinvention, a cell separated as appropriate from an animal or anestablished cell line can be mentioned in the case of cells derived fromanimals. Their examples include a non-small cell lung cancer cell line,an adipose cell, a brain- or nerve-derived cell, a cell line or thelike. As the tissue derived from animals, organs or tissues harvested asappropriate from the animals can be used, and as an example a braintissue section in the form containing the hypothalamus can be used.

The determination of expression intensity of the nesfatin polypeptideetc., or the gene encoding said polypeptide can be carried out by animmunological method or by determining mRNA. In the immunologicalmethod, a format compatible with the purpose of determination or thebiological sample may be used as appropriate. For example, when thesample is a cell derived from an animal, flow cytometry orcytoimmunological staining etc. can be used, and when tissue specimensderived from animals are used, immunohistochemical methods etc. may beselected. When preparations such as lysates from samples are used, theELISA method, the RIA method, the Western blotting method etc. can beselected. When mRNA is to be determined, a format compatible with thepurpose of determination, or the biological sample, may be used asappropriate. When, for example, animal-derived cell samples or tissuesamples are used as they retain the morphology, the in situhybridization method etc. can be selected, and when mRNA extracted fromsaid samples is used, the RT-PCR method, the Northern blotting methodetc. can be selected.

The present invention discloses that the PPARγ agonist enhances theexpression of the nesfatin polypeptide etc. and the gene encoding it.Thus, In the above step of “contacting a candidate compound with amammal-derived cell or tissue”, the compounds that suppresses theexpression of the nesfatin polypeptide etc., or the gene encoding ofsaid polypeptide of which expression is enhanced can be screened bycontacting PPARγ such as troglitazone with the sample simultaneouslywith, before or after contact of the candidate compound with the sample,and the compounds that enhance expression of said polypeptide or thegene encoding it, in a mechanism that does not involve PPARγ, can bescreened.

The compound can also be screened by determining the reaction of thecell that occurs when the nesfatin polypeptide etc. acts on the cell. Asan example of such screening, there can be mentioned those that includethe following steps:

(1) a step of contacting a candidate compound with a mammal-derived cellor tissue;

(2) a step of contacting the nesfatin polypeptide etc. with said cell ortissue sample; and

(3) a step of determining the presence or absence of the reaction of thecell, or intensity, by the nesfatin polypeptide etc., as compared to thecontrol that was not contacted with the candidate compound.

In this case, a compound that enhances the reactive intensity of thecell by the nesfatin polypeptide etc. can be a candidate of atherapeutic agent for suppressing food intake and/or suppressing bodyweight gain. Also, a compound that decreases the reactive intensity ofthe cell by the nesfatin polypeptide etc. can be a candidate of atherapeutic agent for enhancing food intake and/or enhancing body weightgain. Substances having the latter activity include, but are not limitedto, an antibody that binds to the nesfatin polypeptide etc.

As the test cell, a cell separated as appropriate from an animal or anestablished cell line can be mentioned in the case of cells derived fromanimals. The examples include a non-small cell lung cancer cell line, anadipose cell, a brain- or nerve-derived cell, a cell line or the like.As the tissue derived from animals, organs or tissues harvested asappropriate from the animals can be used, and as an example brain tissuesection in a form containing the hypothalamus can be used.

The reaction in the above cell refers to a physical and chemical changeinduced by the action of the nesfatin polypeptide etc. on the cell, andinclude, for example, changes in cell morphology, membrane potential,cell growth, intracellular calcium concentration, migration reaction,intracellular secondary messenger molecule (cAMP, dGMP etc.)concentration and the like.

Furthermore, in the screening method of the present invention, atranscription regulatory region that is in a genome of the nesfatin andthat controls the expression of said gene is obtained, and can be usedin a reporter assay system that employs the nucleic acid molecule ofsaid transcription regulatory region. The reporter assay system refersto an assay system in which a transcription regulatory factor that actson said transcription regulatory region is screened with the amountexpressed of the reporter gene disposed downstream to the transcriptionregulatory region as an index.

As an example of such screening, there can be mentioned those thatinclude the following steps of (1) to (3):

(1) a step of contacting the candidate compound with a cell havingintroduced therein a vector comprising the transcription regulatoryregion of the gene encoding the nesfatin polypeptide and the reportergene under the control of the transcription regulatory region;

(2) a step of determining the activity of said reporter gene; and

(3) a step of selecting a compound that decreases, or a compound thatincreases, the expression level of said reporter gene as compared to thecontrol that was not contacted with the candidate compound.

In this case, a compound that enhances the expression intensity of thereporter gene can be a candidate of a therapeutic agent for suppressingfood intake and/or suppressing body weight gain. Substances having suchan activity include, but are not limited to, a PPARγ agonist and thelike. Also, a compound that decreases the expression intensity of thereporter gene can be a candidate of a therapeutic agent for enhancingfood intake and/or enhancing body weight gain. Substances having such anactivity include, but are not limited to, an antibody that binds to thenesfatin polypeptide etc., an antisense oligonucleotide against the geneencoding said polypeptide, a PPARγ antagonist and the like.

As the transcription regulatory region, there can be mentioned apromoter, an enhancer, a CAAT box commonly seen in the promoter region,or a TATA box. As the reporter gene, there can be used thechloramphenicol acetyltransferase (CAT) gene, the luciferase gene, thegrowth hormone gene and the like.

As the cell into which a reporter gene vector is introduced, there canbe mentioned a cell line separated or established from animals,non-mammalian cells such as yeast, and the like.

As the method of introducing the reporter gene vector into the host,there can be illustrated the biological method, the physical method, thechemical method and the like. As the biological method, there can beillustrated a method that employs a virus vector, a method that employsa specific receptor, cell fusion (Sendai virus (HVJ)), polyethyleneglycol (PEG), electric cell fusion, micronucleate cell fusion(chromosome transfer)), and the like. The physical method includes amicroinjection method, an electroporation method, a method that employsa gene particle gun. The chemical method includes a calcium phosphateprecipitation method, a liposome method, a DEAE-dextran method, aprotoplast method, an erythrocyte ghost method, an erythrocyte membraneghost method, and a microcapsule method.

Polynucleotides, antibodies, cell lines, or model animals required forvarious screening methods of the present invention can be assembled inadvance to make kits. These kits may contain, as additional elements, asubstrate compound for use in labelling, a medium or a vessel forculturing cells, a positive or negative standard sample, andinstructions describing the method of using the kit. The elements may bemixed in advance as needed, and each element may contain preservativesand/or antiseptics as needed.

The present invention will now be explained in more detail hereinbelowwith reference to Working Examples, but it should be noted that theseExamples do not limit the present invention in any way. In the followingWorking Examples, unless otherwise specified, each procedure wasperformed in accordance with the methods described in “MolecularCloning” (Sambrook, J., Fritsch, E. F. and Maniatis, T., issued by ColdSpring Harbor Laboratory Press in 1989), or when commercially availablereagents or kits are used, they were used as described in theinstructions attached in the commercially available products. Inexperiments using animals, unless otherwise specified, animals (malerats) were kept under an illumination cycle of 12 hours of the lightperiod from 6 a.m. to 6 p.m. and 12 hours of the dark period from 6 p.m.to 6 a.m. the next morning at 22° C., and the experiment was carried outunder the authority of the animal experiment facility of GunmaUniversity.

WORKING EXAMPLES Working Example 1 Cloning of the Gene Induced byStimulation with the PPARγ Agonist

In order to identify the gene that acts on food intake control and/orbody weight control among the genes of which expression is regulated byPPARγ, genes that are specifically induced when SQ-5 cells (mainlyderived from humans) are stimulated with the PPARγ agonist were cloned,and from them, genes that are likely to function as a secretary factorwere selected.

The method of obtaining genes of which expression is specificallyinduced by PPARγ in non-small cell lung cancer was carried out accordingto the method of Satoh et al., Oncogene (England), 2002, Vol. 21, pp.2171-2180. The method is briefly described below. From each of SQ-5(Riken BioResource Center RBC0110) cultured for 48 hours understimulation with 10⁻⁴ M of troglitazone (Sankyo Co., Ltd.), a PPARγagonist, and SQ-5 cells not stimulated with troglitazone, poly(A)+RNA(mRNA) was prepared, and was subjected to a reverse transcriptionreaction to prepare double stranded cDNA. A subtraction method wascarried out in which the gene expressing in common cDNA (driver) usingthe cells being expressed in the non-stimulated cells is subtracted fromcDNA (tester) prepared from the cells stimulated with troglitazone usingthe double stranded cDNA obtained. The subtraction method was carriedout using fragments obtained by cleaving the tester and driver cDNA witha restriction enzyme RsaI and by the Clontech's PCR-based cloning kitaccording to the attached protocol. The residual cDNA after subtractionwas subjected to electrophoresis on a 1% agarose gel in TAE buffer (40mM Tris-acetate, 1 mM EDTA), and then cDNA with a length correspondingto 0.5-2.0 kb was collected and ligated to the PGEM®-T Easy vector usingthe PGEM®-T Easy Vector System I (Promega Corp., Cat. No. A1360)according to the attached protocol. The ligated vector was introducedinto a E. coli strain DH5α and allowed to form colonies on an agarmedium prepared with a LB medium containing 50 μg/ml ampicillin, and thecolonies obtained were picked up to obtain clones containing cDNA of thegene of which expression is specifically induced by troglitazonestimulation.

Each clone was identified by analyzing the nucleotide sequence of thecloned cDNA. E. coli of each clone obtained was cultured overnight in a10 mL of the LB medium containing 50 μg/ml ampicillin, and a plasmid wasprepared from the cells using the QIAGEN's QIAprep®Spin Miniprep kitaccording to the attached protocol.

For the plasmid of each clone obtained, a reactant for nucleotidesequence analysis was prepared using the T7 primer (Promega Corp., Cat.No. Q5021) and the BigDye Terminator Cycle Sequencing FS Ready Reactionkit (Cat. No. 4303149) of Applied Biosystems according to the attachedprotocol, and its nucleotide sequence was analyzed using the ABI337 typeDNA Sequencer (Perkin-Elmer).

By subjecting the nucleotide sequence of cDNA of each clone as a queryto the BLAST method with the sequences on the EMBL and the Genbanknucleic acid database, a sequence was obtained for which the full-lengthcDNA sequence was known.

From the full-length cDNA sequence obtained in the above process, cDNAencoding a protein having a signal peptide was analyzed by the Signal Psoftware in order to select secretary factors.

<Result>

Clones obtained by the subtraction of cDNA of the SQ-5 cells notstimulated with troglitazone using the cDNA obtained from the SQ-5 cellsstimulated with troglitazone were 596 clones. For 213 clones among them,remarkable homology was noted with the registered sequences in thenucleic acid sequences in EMBL and Genbank. Furthermore, by the analysisof signal peptides, 9 clones were suggested to have a signal peptidesequence. One of the 9 clones was the human NEFA gene for which afunction had not been identified (SEQ ID NO: 1).

Working Example 2

Induction of the NEFA Gene Expression in a Cultured Cell Line by PPARγAgonist Stimulation

In order to confirm the induction of the NEFA gene expression bytroglitazone, cell lines HTB185 and SQ5 expressing PPARγ and an adiposecell line 3T3-L1 were analyzed for the expression of the NEFA gene byNorthern blotting.

SQ-5 (Riken BioResource Center RBC0110) was subcultured in a RPMI 1640medium (Invitrogen-BRL, Cat. No. 11875-085) containing 10% bovine fetalserum (Invitrogen-GIBCO). Human cerebrospinal blastocytoma cell lineHTB185 cells (D283 Med: ATCC HTB185) and mouse fetal fibroblast cellline (precursor adipose cell line) 3T3-L1 cells (ATCC CL-173) of whichdifferentiation was induced with insulin, dexamethasone and IBMX weresubcultured in a DMEM medium (Invitrogen-GIBCO, Cat. No. 11955-040)containing 10% bovine fetal serum. 10⁶ cells from each cell suspended in10 ml of the medium were plated in a dish with a diameter of 10 cm.After the cells adhered to the substrate, DMSO alone (control) ortroglitazone (Sankyo Co., Ltd.) were added to a concentration of 10⁻⁴ M,10^(−4.5) M or 10⁻⁵ M, and cultured under the condition of 5% CO₂ at 37°C. for 24 hours or 48, hours. For each experiment group, cells treatedin 5 dishes were scraped from the substrate after a certain period, andtotal RNA was extracted and collected using ISOGEN (Nippon Gene, Cat.No. 317-02503) according to the method described in the protocol.

A probe for detecting the NEFA gene was prepared using a 565 bp DNAfragment set forth in SEQ ID NO: 21 by labelling with in vitrotranscription using a SP6 polymerase. Using a plasmid extracted from themouse brain cDNA library (Invitrogen Corp., Cat. No. 10655-25) as thetemplate and the following-primers at a concentration of 0.2 μM,amplified DNA was prepared by 35 cycles of reaction comprisingdenaturation at 94° C. for 1.5 minute, annealing at 58° C. for oneminute and amplification at 72° C. for 2 minutes (Takara Bio Inc.,Takara Ex Taq™ polymerase, Cat. No. RR001A 2.5U was used).

PCR primers for preparing the NEFA probe:

(SEQ ID NO: 22) Forward primer: 5-CCAGTGGAAAATGCAAGGAT-3 (SEQ ID NO: 23)Reverse primer: 5-TCTTTGCTTCCGGGATGATTA-3

After the purity of the amplified DNA product obtained was confirmed bya 2% agarose gel electrophoresis using the TAE buffer, it was subclonedinto the pGEM®-T Easy vector using the pGEM®-T Easy Vector System I(Promega Corp., Cat. No. A1360) according to the attached protocol, andintroduced into the E. coli DH5α to form colonies on an agar mediumprepared with the LB medium containing 50 μg/ml ampicillin. Severalcolonies obtained were each picked up, and the E. coli was culturedovernight in 10 ml of the LB medium containing 50 μg/ml ampicillin. Fromthe cells, plasmid was prepared using the QIAGEN's QIAprep® SpinMiniprep kit according to the attached protocol. A reactant fornucleotide sequence analysis was prepared using the T7 primer (PromegaCorp., Cat. No. Q5021) or the SP6 primer (Promega Corp., Cat. No. Q5011)and the BigDye Terminator Cycle Sequencing FS Ready Reaction kit (Cat.No. 4303149) of Applied Biosystems according to the attached protocol,and its nucleotide sequence was analyzed using the ABI337 DNA Sequencer(Perkin-Elmer). A clone that has a sequence corresponding to thepredicted sequence and in which the 5′-end of the amplified NEFA-derivedgene faces the T7 promoter side of the vector was made a clone forobtaining a plasmid for preparing the probe. The clone obtained wascultured overnight in a 10 mL of the LB medium containing 50 μg/mlampicillin, and a plasmid was prepared from the cells using the QIAGEN'sPlasmid Mini kit according to the attached protocol. The preparedplasmid was cleaved with a restriction enzyme NcoI, extracted withphenol, purified with phenol/CIAA extraction, and after precipitatingwith ethanol, it was dissolved at 1 μg/ml in a ribonuclease-free TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to prepare a template plasmidDNA for preparing the probe. A labelled RNA probe was prepared from 1 μgof the template plasmid DNA using Promega's Riboprobe® System-SP6 with2.5 μM [α-³²P]UTP (Amersham Biosciences Inc., Cat. No. PB10203, 3000Ci/mM, 10 mCi/ml) and 20U of RNA synthase SP6 (contained in the kit)according to the protocol attached to the kit.

For electrophoresis for Northern blotting, total RNA obtained from eachcell was subjected to electrophoresis using a 1.2% agarose gel preparedin a MOPS buffer (20 mM 3-(N-morpholino)-propanesulfonic acid, 5 mMsodium acetate, 0.5 mM EDTA) containing 0.66 M deionized formaldehyde at100 V for 2 hours. Using a 10-fold concentration of SSC (150 mM NaCl, 15mM sodium acetate), RNA was transferred from the gel afterelectrophoresis to a nylon membrane (Perkin-Elmer Inc., Gene ScreenPlus® Hybridization Transfer Membrane). To the transferred membrane, RNAwas immobilized by UV using Stratagene's Stratalinker. Then, the nylonmembrane was subjected to prehybridization in a prehybridizationsolution (0.25 M NaCl, 20 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, 1% SDS, 0.5%bovine serum albumin, 0.5% polyvinyl pyrrolidone, 0.5% Ficoll, 50%deionized formamide) at 43° C. for 3 hours, and to hybridization in ahybridization solution (0.25 M NaCl, 20 mM Tris-HCl, pH 7.5, 2.5 mMEDTA, 1% SDS, 0.5% bovine serum albumin, 0.5% polyvinyl pyrrolidone,0.5% Ficoll, 50% deionized formamide, 10% dextran sulfate) containing1,000,000 cpm/ml of the labelled RNA probe at 43° C. overnight. Afterrinsing the nylon membrane after hybridization with a two-foldconcentration of SSC, it was washed twice under a stringent condition of0.2-fold concentration of SSC containing 0.1% SDS at 60° C. for 30minutes. The nylon membrane after washing was exposed overnight to anX-ray film (Kodak, XAR film) in the presence of an intensifying screen(Cronex, Lightining Plus), and the labelled RNA probe that hybridizedwas detected.

<Result>

The result of Northern blotting analysis of the induction of the NEFAgene with troglitazone (TGZ) in a cell line is shown in FIG. 1. Lanes1-4 represent results for the human medulloblastoma cell line HTB185cells that are expressing PPARγ. Lanes 1 and 3 show the detection of theNEFA gene expression with the RNA of cells cultured (24 hours and 48hours, respectively) without the TGZ stimulation of the HTB185 cells,while lanes 2 and 4 show the detection of the NEFA gene expression withthe RNA of cells stimulated 10⁻⁴ M troglitazone for 24 and 48 hours,respectively. As a result, only a slight expression of the NEFA gene wasnoted in the cells not stimulated with TGZ (lanes 1 and 3), whereasmarked induction was noted in the cells stimulated with TGZ for 48 hours(lane 4). Lanes 5 and 6 in the Figure represent the result obtained withSQ-5 that is expressing PPARγ. Lane 5 shows the detection of the NEFAgene expression with the RNA of the SQ-5 cells cultured without TGZstimulation, while lane 6 shows the detection of the NEFA geneexpression with the RNA of cells stimulated 10⁻⁴ M TGZ for 24 hours. Asa result, a low level of expression was noted in the cells notstimulated with TGZ (lane 5), whereas marked induction of expression ofthe NEFA gene was noted in the cells stimulated with TGZ for 24 hours(lane 6). Furthermore, lanes 7-10 in FIG. 1 represent results for themouse fetal fibroblast cell line (precursor adipose cell line) 3T3-L1cells that are expressing PPARγ. Lane 7 shows the detection of the NEFAgene expression with the RNA of the 3T3-L1 cells cultured without TGZstimulation, while lanes 8-10 show the detection of the NEFA geneexpression with the RNA of the 3T3-L1 cells stimulated with 10⁻⁵ M,10^(−4.5) M and 10⁻⁴ M of TGZ, respectively, for 48 hours. As a result,a considerable amount of expression of the NEFA gene was noted even inthe absence of TGZ stimulation (lane 7), and similar expression wasobtained when stimulated with 10⁻⁵ M to 10⁻⁴ M of TGZ, indicating thatthe NEFA gene is being constantly expressed irrespective of thestimulation of PPARγ.

Working Example 3 Preparation of Antibody Against NEFA and theDemonstration of Expression in the Rat Brain

The secretary protein expressed in the adipose cells is also expressedin the brain, and thus has been proposed as the Brain-Adipose Axis thatis likely to have an effect of controlling food intake (Masatomo Mori:Adipose Research (Shibou Kenkyu), 2004, Vol. 10, pp. 117-119, Shimizu H.and Mori M.: Nutritional Neurosci 8:7-20, 2005). Thus, an antibodyagainst NEFA that is expressed in the adipose cells and the brain tumorcells to investigate the localized expression of NEFA in thehypothalamus.

For the preparation of the antibody against NEFA, a synthetic peptide(SEQ ID NO: 24: synthesized at ATP K.K.) in which Cys was attached tothe C-terminal of a sequence from His at position 141 to Ser at position152 of the amino acid sequence (SEQ ID NO: 8) of the rat precursor NEFApolypeptide (said peptide is hereinafter referred to as the NAP peptide)was used as the antigen. NAP peptide:N-HisLeuAsnHisGlnAsnProAspThrPheGluSerCys-C (SEQ ID NO: 24).

Said synthetic NAP peptide was conjugated to keyhole limpet hemocyanin(KLH) using the Imject® Maleimide Activated Mariculture Keyhole LimpetHemocyanin of Pierce Inc. according to the attached protocol. 0.2 mg ofthe peptide obtained was used for one immunization per rabbit. Forimmunization, 0.25 ml of the conjugate solution (conjugateconcentration: 1 mg/ml) and an equal amount of Freund's completeadjuvant H-37 Ra (Wako Pure Chemical—Difco, Cat. No. 528-00031) weremixed, and 50 μl each was intradermally injected at 8 different placeson the shaved back of New Zealand White rabbits (purchased from ImaiExperimental Animal Testing Site). Similar immunization was performedfurther 4 times every two weeks. One week after the last immunization,part of the blood was collected, and antibody titer in the serum wasconfirmed by the ELISA method employing the immobilized peptideconjugate. On the next day, the animals were sacrificed to collect wholeblood. From the blood obtained, serum was prepared. From the serumrabbit IgG was purified using the DEAE Sepharose FF (AmershamBiosciences Inc., Cat. No. 17-0709-10) according to a standard method.The purified rabbit IgG was affinity-purified using apeptide-immobilized column that was prepared from 1 mg of the NAPpeptide using the SulfoLink kit (Pierce Inc., Cat. No. 44895), accordingto the protocol attached to the kit.

Using the obtained antibody (anti-NAP polyclonal antibody) against theNAP peptide, the expression of the NEFA polypeptide in the rat brain wasanalyzed by Western blotting.

Protein extracts from the rat brain was prepared by homogenizing thehypothalamus (1.7 g) of 8 week-old Wistar rats (Nippan Charles River) in5 ml of the extraction buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mMEDTA, 1% Nonidet P-40). The rat brain lysate obtained was mixed with anequal amount of the Laemmli sample buffer (Bio-Rad Inc., Cat. No.161-0737) containing 5% β-mercaptoethanol for SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and heated to 100° C. for 5 minutes, and thencentrifuged in a microcentrifuge at an ordinary temperature at 15,000rpm for 10 minutes to collect the supernatant. 20 μl of the supernatantwas electrophoresed on a polyacrylamide gel (Bio-Rad Inc., Ready gel10-20%, Cat. No. 161-J390V) using the Tris-glycine/SDS buffer (25 mMTris, 192 mM glycine, 0.1% SDS, pH 8.3) at 100V for 1 hour, and thentransferred to a nitrocellulose membrane. The membrane was blocked in 3%gelatin/TBS at room temperature for 1 hour, and washed three times inTBS-0.05% Tween and once in TBS. It was then allowed to react overnightat room temperature in a 1% gelatin/TBS solution containing 1 μg/ml ofanti-NAP polyclonal antibody. On the next day, it was washed three timesin TBS-0.05% Tween and once in TBS, and then was reacted withanti-rabbit IgG goat polyclonal antibody-peroxidase conjugate (CappelInc., Cat. No. 674371) diluted to about 1 μg/ml in 1% gelatin/TBSsolution at room temperature for 1 hour. It was then washed for threetimes in TBS-0.05% Tween and twice in TBS, and was allowed to developcolor using the HRP color development kit (Bio-Rad Inc., Cat. No.170-64631) at room temperature for five minutes, and the bands weredetected

<Result>

FIG. 2 A shows the schematic diagram of the structure of the NEFApolypeptide and the position of the peptide, which was used forpreparing the antibody, on NEFA, and FIG. 2 B shows the result ofWestern blotting of the protein extracts from the rat brain with apolyclonal antibody prepared by immunization with the NAP peptide. Inthe Western blotting of the protein extracts from the rat brain with apolyclonal antibody prepared against the NAP peptide, a single band wasobserved at a molecular weight of about 47.5 kd (FIG. 2 B). Thisrevealed that the antibody against NAP recognizes the full-length NEFAand that NEFA is being expressed in the rat brain.

Working Example 4 Study on the Expression Site of nesfatin in the RatBrain Hypothalamus

In order to analyze the expression site of NEFA in the rat brainhypothalamus associated with the control of food intake, animmunohistochemical analysis of the sections of the rat brain wascarried out. Eight week-old Wistar rats (purchased from Nippon SLC) wereanesthetized by an intraperitoneal injection of 40 mg/kg ofpentobarbital sodium, and then thoracotomy was performed to inject 50 mlof physiological saline (0.85% NaCl) into the heart, and then PBScontaining 4% paraformaldehyde (20 mM phosphate buffer, 150 mM NaCl, pH7.2) was injected and circulated using a 400 ml peristatic pump toperfusion fix the brain. The brain was then extracted, and the portioncontaining the hypothalamus was excised, and was fixed overnight at 4°C. in PBS containing 4% paraformaldehyde. The fixed brain sample wasimmersed in PBS containing 10% sucrose for 4 hours, PBS containing 10%sucrose for 4 hours, and PBS containing 20% sucrose overnight at 4° C.Then the brain sample was immersed in an OCT compound and frozen in dryice-acetone to prepare the embedded block. From the block prepared,sections of 6 μm in thickness were prepared at −20° C. using a cryostat,and then were air-dried on a silane-coated slide (MAS coat slideprepared by Matsunami). Tissue sections on the slide were treated with3% hydrogen peroxide for 5 minutes, and then washed twice in PBS for 5minutes. Furthermore, the tissue sections were blocked by treating with10% goat normal serum (Nichirei Corp., Cat. No. 426042) at roomtemperature for 10 minutes, and then were reacted to 1 μg/ml of apolyclonal antibody (Working Example 3) against the NAP peptide dilutedin PBS containing 0.5% BSA at room temperature for 1 hour (Ab group).After washing the slide in PBS, the slide was reacted with the HistofineSimple Stain Rat MAX PO (Nichirei Corp., Cat. No. 414181), and afterthree times-washing in PBS for 5 minutes per each, color was developedusing the Simple Stain DAB solution (Nichirei Corp., Cat. No. 415172).The slide after color development was washed in water, and then stainedwith the Meyer-Hematoxylin (Muto Pure Chemicals Co., Ltd., Cat. No.3000), washed in water, dehydrated in alcohol and cleared in xylene, andmounted using a non-aqueous mounting agent (Nichirei Corp., Cat. No.415141) with a cover slip. The brain section on the slide after mountingwas examined with a microscope.

<Result>

FIG. 3 shows a micrograph (200× magnification) of histoimmunochemicalstaining with the anti-NAP peptide in the tissue section at thehypothalamus and the third ventricle (3V) regions of the rat's brain.Specific staining was seen in Arc: arcuate nucleus, PVN: paraventricularnucleus, LH: lateral hypothalamic area, SON: supraoptic nucleus of thehypothalamus in the figure, indicating the expression of NEFA in thesesites. From the foregoing, it was demonstrated that the NEFA polypeptideis expressed in the rat hypothalamus, specifically in the arcuatenucleus, the paraventricular nucleus, the lateral hypothalamic area andthe supraoptic nucleus.

Since the expression of the NEFA polypeptide encoded by the NEFA genewas noted at sites associated with the control of food intake in thehypothalamus, said polypeptide was named nesfatin.

Working Example 5 Preparation of Recombinant nesfatin

In order to confirm the properties of nesfatin expressed in the nervenucleus of the hypothalamus associated with food intake, recombinantnesfatin was prepared in E. coli. The recombinant matured mouse nesfatin(SEQ ID NO: 26) was purified by expressing a fusion protein (recombinantmouse GST-NEFA: SEQ ID NO: 25) in which glutathione S-transferase (GST)was bound to the N-terminal of mouse matured mouse nesfatin (SEQ ID NO:6), and treating the fusion protein with a protease. The level ofhomology on the amino acid level of the rat nesfatin and the mousenesfatin is as high as 96.5%.

A gene for expressing recombinant mouse GST-NEFA was prepared using aplasmid extracted from the mouse brain cDNA library (Invitrogen Corp.,Cat. No. 10655-25) as the template and using the primers described below(0.25 μM each) in a denaturation reaction at 94° C. for 1.5 minute,followed by five cycles of reaction comprising denaturation at 94° C.for 0.5 minute, annealing at 60° C. for 0.5 minute, and extension at 72°C. for 2 minutes and then 30 cycles of reaction comprising denaturationat 94° C. for 0.5 minute, annealing and extension at 72° C. for 2minutes, to prepare the amplified DNA (Stratagene's PfuTurbo®polymerase, Cat. No. 600250 was used).

PCR primers for preparing recombinant matured mouse NEFA cDNA

Forward primer: (SEQ ID NO: 27) 5-TTGGATCCGTTCCTATCGATGTGGACAAGAC-3Reverse primer: 5 (SEQ ID NO: 28) 5-TTGCGGCCGCTTATGTGTGTGGCTCAAACTTCAG-3

The PCR product obtained was purified by QIAGEN's MinElute PCRPurification Kit, cleaved with restriction enzymes BamHI and NotI, andan about 1.3 kb band was excised by a 2% agarose gel-TAE electrophoresisand the DNA fragment was collected (QIAGEN's MinElute Gel ExtractionKit, Cat. No. 28604 was used). The 1.3 kb DNA fragment was ligated topGEM-11Zf(+) vector (Promega, P2411) that had been cleaved withrestriction enzymes BamHI and NotI, which was transformed to E. colistrain DH5α (Takara Bio Inc., Cat. No. 9057) and was allowed to formcolonies on an agar medium comprising an LB medium containing 50 μg/mlampicillin.

Several of the colonies obtained were picked up, and for the plasmidprepared from each clone the nucleotide sequence was analyzed usingpUC/M13 forward primer (Promega Corp., Cat. No. Q5391), pUC/M13 reverseprimer (Promega Corp., Cat. No. Q5401), two kinds of primers mSQ1(5-CCTGAACCACCAGAATCC-3: SEQ ID NO: 29) and mSQ2(5-AGACTGATGGATTGGACC-3: SEQ ID NO: 30) in the sequence of the mouseNEFA gene, and using the BigDye Terminator Cycle Sequencing FS ReadyReaction kit (Cat. No. 4303149) of Applied Biosystems and the ABI337type DNA Sequencer (Perkin-Elmer). Clones that were shown to have anucleotide sequence corresponding to SEQ ID NO: 11 by the nucleotidesequence analysis were cultured overnight in 10 ml of the LB mediumcontaining 50 μg/ml ampicillin, and plasmid was prepared from the cellsusing the QIAGEN's QIAprep®Spin Miniprep kit according to the attachedprotocol.

The plasmid was cleaved with restriction enzymes BamHI and NotI, waselectrophoresed on a 2% agarose TAE gel, and the 1.2 kb band was excisedto collect DNA. The collected DNA fragments were ligated to thepGEX-4T-1 vector (Amersham Biosciences Inc., Cat. No. 27-4580-01) thathad been cleaved with restriction enzymes BamHI and NotI, and which wastransformed to E. coli strain BL21 (Amersham Biosciences Inc., Cat. No.27-1542-01) using the Gene Pulser Xcell electroporation system (Bio-RadInc., Cat. No. 165-2660J1) so as to allow colonies to be formed on anagar medium prepared with the LB medium containing 50 μg/ml ampicillin.Recombinant E. coli obtained by transformation was confirmed for thepresence or absence of an insert by PCR using the pGEX5′ SequencingPrimer and the pGEX3′ Sequencing Primer (Amersham Biosciences Inc., Cat.No. 27-1410-01 and 27-1411-01), and the insertion of the NEFA gene wasconfirmed by PCR using the PCR primer set for preparing the NEFA probeused in Working Example 2. Clones for which the matured NEFA cDNA wasconfirmed to be integrated into the pGEX-4T-1 vector were used toexpress the recombinant mouse GST-NEFA protein. The clones were culturedovernight at 37° C. with 10 ml of a LB medium containing 100 μg/mlampicillin, and 5 ml of it was added to 500 ml of a LB medium containing100 μg/ml ampicillin and cultured by shaking at 37° C. until theabsorbance at a wavelength of 600 nm became 0.6 (some of the bacteriawere sampled out: Preinduced bacteria). To the medium was addedisopropyl-β-D-thiogalacto-pyranoside (IPTG: Gibco BRL) to a finalconcentration of 1 mM, and was further cultured by shaking at 37° C. for3 hours. After culturing, the medium containing the cells werecentrifuged at 5,000 g at 4° C. for 15 minutes to collect the cells,which were suspended in 25 ml of PBS. The suspension was frozen at −80°C. for about 1 hour, and then thawed quickly at 37° C., and the cellswere subjected to sonication for 30 seconds× five times to disrupt thecells. It was followed by centrifugation at 10,000 g at 4° C. for 30minutes to collect the supernatant (the precipitated fraction wassampled out: Post-sonicated pellet).

The supernatant was applied to a 5 ml-scale GSTrapFF column (AmershamBiosciences Inc., Cat. No. 17-5131-01) equilibrated with PBS using theAKTA-FPLC liquid chromatography system (Amersham Biosciences Inc.), andthen the column was washed with 50 ml of PBS (flow rate: 1 ml/min). Thenthe elution buffer (10 mM reduced glutathione, 0.4 M Tris-HCl, pH 8.0,0.2 M NaCl) was run at a flow rate of 1 ml/min to collect 1 mlfractions, and fractions containing protein as determined by absorbanceat a wavelength of 280 nm were collected (the fraction having thehighest absorbance at 280 nm: Purified GST-NEFA). A portion of each ofthe Preincubation bacteria fraction, the Post-sonicated pellet fraction,and the Purified GST-NEFA fraction was dissolved in the Laemmli samplebuffer (Bio-Rad Inc., Cat. No. 161-0737) containing 5%β-mercaptoethanol, treated at 100° C. for 5 minutes, and centrifuged ina microfuge at 15,000 rpm for 5 minutes to obtain a supernatant, whichwas subjected to a polyacrylamide gel electrophoresis (Bio-Rad Inc.,Ready gel 10-20%, Cat. No. 161-J390V) at 100 V for 1 hour using aTris-glycine/SDS buffer and then stained in a Coomassie brilliant bluestain (Bio-Rad Inc., Bio-Safe CBB G-250, Cat. No. 161-0786). For most ofthe Purified GST-NEFA fraction, reduced glutathione was removed from thesample using liquid chromatography with the HITrap Desalting column(Amersham Biosciences Inc., Cat. No. 17-1408-01) equilibrated with PBS,and the sample was applied again to a 5 ml-scale GSTrapFF columnequilibrated with PBS. After the column was washed with 25 ml of PBS ata flow rate of 1 ml/min, 15 ml of the protease reaction buffer (50 mMTris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) was run into thecolumn, and 4.6 ml of the protease reaction buffer containing 20 U/mlthrombin (Amersham Biosciences Inc., Cat. No. 27-0846-01) was applied tothe column. When the entire volume entered the column, the pump of theliquid chromatography was stopped, and reaction with protease wascarried out at room temperature (22° C.) overnight. On the next day, 15ml of PBS was run through the column at a flow rate of 1 ml/min tocollect fractions, and the fractions collected were run through theHiTrap Benzamidine FF column (Amersham Biosciences Inc., Cat. No.17-5143-02) to obtain a fraction in which thrombin was removed(Prepurified sample). The fraction was fractionated by chromatographywith an anion exchange resin to purify the recombinant matured mouseNEFA polypeptide. The Prepurified sample (20 ml) was diluted 5-fold withthe Buffer A (20 mM bis-Tris, pH 6.5, 0.1 M NaCl, 0.1% CHAPS), andapplied to the HiTrap Q HP column (Amersham Biosciences Inc., Cat. No.17-1153-01) equilibrated with the Buffer A, and then washed with theBuffer A at a flow rate of 1 ml/min (1 ml fractions were collected:Washed sample). The washed column was eluted using the Buffer A and theBuffer B (20 mM bis-Tris, pH 6.5, 1M NaCl, 0.1% CHAPS) in a linearconcentration gradient of 0-50%/60 minutes of the Buffer B at a flowrate of 0.5 ml/min. At this time, 0.5 ml fractions were collected, andfractions containing protein as determined by absorbance at a wavelengthof 280 nm were collected (Purified sample). A portion of each of thesamples (Prepurified sample, Washed sample, Purified sample) obtained inthis purification process was taken, and was mixed with an equal amountof the Laemmli sample buffer containing 5% β-mercaptoethanol followed byheating at 100° C. for 5 minutes, and then was subjected to Westernblotting in a manner similar to that described in Working Example 3. Thefraction was dialyzed against PBS to obtain a purified matured mouseNEFA polypeptide.

<Result>

A of FIG. 4 shows a stained image of SDS-PAGE of a sample obtained byexpressing the GST-NEFA fusion protein in recombinant E. coli andpurifying the GST-NEFA fusion protein with the GSTrapFF column. B showsthe result of Western blotting using anti-NAP polyclonal antibody of asample obtained by excising the matured mouse NEFA polypeptide from theGST-NEFA fusion protein with thrombin and purifying with the HiTrap Q HPcolumn. In A of FIG. 4, a marked increase in the product with amolecular weight 65 kd was noted in the Post-sonicated pellet whereinthe cells were disrupted after the expression induction with IPTG inlane 3 relative to the Preincubated bacteria sample in lane 2. As themajor 65 kd product was also noted in the fraction (Purified sample)purified with the GSTrapFF column, it was demonstrated that therecombinant mouse GST-NEFA fusion protein was expressed in therecombinant E. coli. In B of FIG. 4, several bands were noted in thesample before purification (Prepurified sample) in addition to the bandof the matured mouse NEFA polypeptide with a molecular weight of 47.5 kdcleaved with thrombin and the band of the recombinant mouse GST-NEFAfusion protein with a molecular weight of 65 kd. On the other hand, inthe Purified sample, a major band with a molecular weight of 47.5 kd anda weaker band with a molecular weight of 65 kd were noted, indicatingthat an almost pure recombinant matured mouse NEFA polypeptide wasobtained.

Working Example 6 Study on the Effect of Intraventricular Administrationof nesfatin on Feeding Behavior

In order to demonstrate the effect of recombinant nesfatin obtained onthe feeding behavior of animals, an experiment was carried out in whichunanesthetized rats received intra-cerebroventricular administrationinto the third ventricle.

Rats used were Wistar rats (purchased from Nippon SLC), and the animalswere housed under a cycle of 12 hours of the light period from 6 a.m. to6 p.m. and 12 hours of the dark period from 6 p.m. to 6 a.m. the nextmorning and fed a powder chew (Nippon Clea, CE-2), and kept at 22° C.,and housing was continued under a similar condition during theexperiment. For the experiment, among 8-9 week-old male Wistar rats,those individuals weighing 200-250 g were used. For administration, 5 μlof saline (control) or recombinant nesfatin prepared in Working Example5 dissolved to 0.2 pmol, 0.6 pmol and 3.0 pmol in 5 μl of PBS wasadministered per individual (N=5 for each group). The rats receivedintraperitoneal administration of 40 mg/kg body weight of pentobarbitalsodium for anesthesia. After the hair was removed from the head, theywere fixed in a brain stereotaxic apparatus (David Kopf Instruments,Model 962) and the scalp was incised and a 23G needle (guide cannula)was implanted so as to reach the third ventricle (2.5 mm behind thebregma, 9.5 mm from the surface). One week after implanting the guidecannula, the animals were subjected to the experiment. After recoveringfrom the surgery, a 29G cannula for injection was inserted into theguide cannula under no anesthesia, and 5 μl of PBS or the matured mousenesfatin solution in PBS was administered at a rate of 1 μl/second.After fixing the cannula as it was for 2-3 minutes after theadministration, the cannula was extracted, and the animals were returnedto the individual cages. One, three, and six hours thereafter, theweight of the food was determined to record the amount of food intake,which was used as an index of feeding behavior. For the test ofsignificant difference, analysis of variance was used.

<Result>

The amount of food intake at 0-1 hour after the administration of 0 (PBSalone), 1, 4 and 20 pmol of recombinant nesfatin into the thirdventricle is graphically shown in A of FIG. 5, the amount of food intakeat 1-3 hours after administration is graphically shown in B of FIG. 5,and the amount of food intake at 3-6 hours after administration isgraphically shown in C of FIG. 5. For any of 0-1, 1-3 and 3-6 hours, areduction in the amount of food intake was noted in a dose dependentmanner in the recombinant nesfatin administration group compared to therats that only received saline. Specifically, in the 20 pmol recombinantnesfatin-administration group, a significant activity of suppressingfood intake was noted in any of 0-1 hour/1-3 hours/3-6 hours (eachP<0.01) as compared to the control group. These data suggested thatnesfatin in the brain has an effect of suppressing feeding behavior.

Working Example 7 Study on the Effect of Intraventricular Administrationof Anti-nesfatin Antibody on Feeding Behavior

In order to demonstrate the effect of nesfatin on the control of foodintake, the effect of administration of an antibody against nesfatininto the third ventricular on feeding behavior was investigated.

The antibody used was the anti-NAP peptide antibody (NAP IgG) preparedin Working Example 4, and 5 μl of antibody diluted in saline wasadministered to rats (dose 5 μg). As the control, the same amount andconcentration of IgG (control IgG) purified from non-immunized rabbitswas administered. Rats used were Wistar rats (purchased from NipponSLC), and were housed as in Working Example 6. Among 8-9 week-old maleWistar rats, those individuals weighing 200-250 g were selected. Thetime of administration was before beginning the light period when thereis little feeding behavior, and the method of administration was similarto that in Working Example 6. During the period of 0-3 hours, 3-6 hours,or 6-9 hours after the intra-celebroventricular administration, theweight of the powder food was determined. For the test of significantdifference, analysis of variance was used.

<Result>

For rats that received the intraventricular administration of controlIgG or the anti-nesfatin antibody (NAP IgG), the amount of food intakeat 0-3 hours after the administration is graphically shown in A of FIG.6, the amount of food intake at 3-6 hours after administration isgraphically shown in B of FIG. 6, and the amount of food intake at 6-12hours after administration is graphically shown in C of FIG. 6. It wasdemonstrated that in individuals that received theintra-celebroventricular administration of anti-nesfatin antibody, foodintake during 0-3 hours (Fig. A of 6) and 3-6 hours (B of FIG. 6) waspromoted in a statistically significant manner (each P<0.001). Thefeeding amount of the rat from the anti-nesfatin antibody-administrationgroup increased about 9 times during 0-3 hours (A of FIG. 6), and about10 times during 3-6 hours, respectively, compared to that of the controlIgG-administration group during the corresponding periods. However,since the period of 6-9 hours was in the dark period and food intake wasstarted in the control IgG-administration group, no significantdifference was noted. Thus, as the administration of the anti-nesfatinantibody promoted food intake by rats, it was demonstrated that theanti-nesfatin antibody suppresses the effect of endogenous nesfatin andthat nesfatin is involved in feeding behavior.

Working Example 8 Study on the Expression of the NEFA Gene in the RatHypothalamus and the Effect of Fasting on the Expression

An in situ hybridization was carried out to determine the effect offasting on changes in the expression of endogenous NEFA gene.

The probe used for in situ hybridization in the brain tissue was theplasmid for preparing the NEFA probe that was prepared in WorkingExample 2. Using 1 μg of said plasmid cleaved with a restriction enzymeNcoI and purified, a DIG-labelled cRNA probe was prepared by a reactionof the SP6-RNA transcriptase using the digoxygenin (DIG) RNA labellingkit (Roche Diagnostics K.K., Cat. No. 1175025). The reaction with theSP6-RNA transcriptase was carried out at 40° C. for 2 hours. After thereaction, 2 μl of 0.2 M EDTA (pH 7.0) was added to 20 μl of the reactionmixture to stop the reaction. After 2.5 μl of 3M LiCl and 75 μl ofethanol cooled to −20° C. were added thereto and mixed, it was allowedto stand at −20° C. overnight. On the next day, the reaction mixture wascentrifuged in a microfuge at 4° C. and 15,000 rpm for 20 minutes andthe supernatant was removed. After adding 50 μl of 70% ethanol to theprecipitate and centrifuging again, the supernatant was removed and theprecipitate was dried. The precipitate was dissolved in the DEPC-treatedwater (Nippon Gene K.K., Cat. No. 314-90205), and the amount of RNA wasdetermined by agarose gel electrophoresis, and was diluted in theDEPC-treated water to a RNA concentration of about 0.1 mg/ml. In orderto shorten the length (about 450 bases) of the cRNA probe obtained, 10μl of the DEPC-treated water and 40 μl of carbonate buffer (60 mM sodiumcarbonate, 40 mM sodium bicarbonate, pH 10.2) were added to 10 μl of theRNA solution at 0.1 mg/ml, and reacted at 60° C. for 30 minutes. To thereaction mixture, 60 μl of the neutralization solution (3 M sodiumacetate, 1% acetic acid, pH 6.0) was added, and 360 μl of ethanol thathad been cooled to −20° C. was further added and mixed, and thencentrifuged in a microfuge at 4° C. and 15,000 rpm for 20 minutes, whichwas precooled at −70° C. for 30 minutes to be set at 4° C., and thesupernatant was removed to obtain the precipitate. After adding 100 μlof 70% ethanol (−20° C.) to the precipitate and centrifuging again, thesupernatant was removed and the precipitate was dried. The driedprecipitate was dissolved in 100 μl of the DEPC-treated water, and theconcentration was determined by agarose gel electrophoresis. Theconcentration of the probe obtained was about 50 μg/ml. The DIG-labelledNEFAcRNA probe thus obtained was used for in situ hybridization.

About 8 week-old male Wistar rats (purchased from Nippon SLC) (bodyweight 220-250 g) were grouped into the individuals (Normal) that cantake the powder food ad libitum, the individuals (Starvation) that werekept 48 hours with drinking water alone without any food, theindividuals (Re-feeding) that were kept without food for 36 hours andthen were given the food ad libitum for 12 hours, and each brain wasperfusion fixed with PBS containing 4% paraformaldehyde in a methodsimilar to that described in Working Example 5 and the brain wasextracted. From the extracted brain, a portion that contains thehypothalamus was excised, which was further fixed in PBS containing 4%paraformaldehyde at 4° C. overnight. The fixed brain tissue was treatedwith a cryostat in a method similar to that described in Working Example4 to prepare sections of 10 μm in thickness, which were placed on asilane-coated glass slide. The slide glass having a brain tissue sectionplaced thereon was placed in an incubator and treated at 50° C. for 2minutes, and then the sections were dried at room temperature for 30minutes. The sections were fixed again in PBS containing 4%paraformaldehyde at room temperature for 7 minutes, and then washed inPBS for 3 minutes and in 2-fold concentrated SSC twice for 5 minutes.

An in situ hybridization solution (4-fold concentrated SSC, 10% dextransulfate, 1-fold concentrated Denhardt's solution, 2 mM EDTA, 50%deionized formamide, 500 μg/ml trout sperm DNA) was delivered so as tocover the brain tissue section on the slide glass, and was prehybridizedat 37° C. for 1 hour. After the completion of prehybridization, thedelivered liquid was discarded, and an in situ hybridization solutioncontaining 200 ng/ml of DIG-labelled NEFA cRNA probe was delivered so asto cover the tissue, and was hybridized in a humid chamber at 37° C. for16 hours. The slide glass having the brain tissue section placed thereonafter hybridization was washed in 2-fold concentrated SSC at 37° C. for5 minutes, and then washed at 37° C. three times for 5 minutes each in0.2-fold concentrated SSC containing 60% formamide, and then at roomtemperature twice for 5 minutes each in 2-fold concentrated SSC.

The probe that hybridized in the brain tissue after washing was detectedusing the DIG Nucleic Acid Detection kit (Roche Diagnostics K.K., Cat.No. 11175041), and the outline of the method is as follows. The slideglass having the brain tissue placed thereon was washed in 100 mMTris-HCl (pH 7.5) buffer containing 150 mM NaCl at room temperature for5 minutes, and blocked using the same buffer (blocking buffer) saturatedwith the blocking reagent contained in the kit at room temperature for30 minutes. On the slide glass was placed anti-DIG-alkaline phosphataseconjugate contained in the kit diluted to a concentration of 1/200 inthe blocking buffer, and it was reacted at room temperature for 2 hours.The slide glass after the reaction was washed twice in 100 mM Tris-HCl(pH 7.5) buffer containing 150 mM NaCl for 5 minutes, and then washed inthe detection buffer (100 Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM MgCl₂)for 10 minutes. Then, the detection buffer containing 0.18 mg/ml BCIPand 0.34 mg/ml NBT was placed and was reacted at room temperature for 16hours to develop color. The slide glass was washed in 10 mM Tris-HCl (ph8.0) containing 1 mM EDTA for minutes to stop the color developmentreaction.

Then the slide glass was washed in distilled water for 5 minutes, andstained in 1% methylene green for 5 minutes, washed in distilled water,dehydrated in alcohol and cleared in xylene, and mounted using anon-aqueous mounting agent (Nichirei Corp., Cat. No. 415141) with acover slip. It was then examined with a microscope.

<Result>

Relative to the control group shown in A of FIG. 7, there was a markeddecrease in the expression of nesfatin mRNA in the paraventricularnucleus (PVN) in the case of 48-hour fasting shown in B of FIG. 7, andas shown in C of FIG. 7, it was demonstrated that the expression of thenesfatin mRNA gene was restored by re-feeding after fasting. Theseresults revealed that endogenous nesfatin is involved in the control offood intake.

Working Example 9 Study on nesfatin Expression in the Brain of StarvatedRats

Changes in endogenous nesfatin at fasting were studied by theimmunohistological stain method.

About 8 week-old male Wistar rats (purchased from Nippon SLC) (bodyweight 220-250 g) were grouped into the control group that can take thepowder food ad libitum and the fasting group that was fasted for 24hours, and the brain tissue section of these rats were prepared for theanalysis by immunohistological chemistry using anti-nesfatin antibody.The preparation of tissue sections and the immunohistological stainmethod were carried out in a manner similar to that described in WorkingExample 4. Furthermore, in order to study the state of activation ofnerve cells in the regions of the brain related to food intake, theimmunohistochemical analysis was carried out at the brain tissue of thefasting group using an antibody against c-Fos. The method was carriedout using a 500-fold diluted c-Fos (K-25) antibody (Santa CruzBiotechnology, sc-253) instead of anti-nesfatin antibody in a mannersimilar to that described above.

<Result>

A micrograph (200× magnification) of immunohistochemical staining withthe anti-nesfatin antibody (NAP Ab) at the hypothalamus of the controlgroup is shown in A of FIG. 8, a micrograph (the same magnification asA) of immunohistochemical staining with the anti-nesfatin antibody (NAPAb) at the hypothalamus of the fasting group is shown in B of FIG. 8,and a micrograph (the same magnification as A) of immunohistochemicalstaining with the anti-c-Fos antibody at the hypothalamus of the fastinggroup is shown in C of FIG. 8. The upper half of the figure shows theimage of the paraventricular nucleus, and the lower half shows the imageof the arcuate nucleus. In the hypothalamus of the fasting group ascompared the control group, staining properties at the paraventricularnucleus (upper half of the figure) and the arcuate nucleus (the lowerhalf of the figure) are markedly decreased, indicating that theexpression of nesfatin is decreased in fasting. In a state in whichappetite is enhanced, the expression of the c-Fos protein was noted.From this, it is believed, in a state in which appetite is enhanced dueto fasting, the expression of nesfatin, thought to exhibit the effect ofa suppressed appetite, is decreased and is responsible for the controlof appetite.

Working Example 10 Preparation of nesfatin-1, nesfatin-2, nesfatin-3 andnesfatin-2/3 polypeptide, and Preparation of Antibody Againstnesfatin-1, nesfatin-3 and nesfatin-2/3 peptide

In Working Examples 3-9, it was demonstrated that nesfatin has an effectthe expression in the brain hypothalamus and on food intake control andbody weight control. Also, as the precursor of nesfatin has a signalpeptide, nesfatin is likely to be secreted extracellularly out of thecell. Some active proteins are known to be cleaved with the prohormoneconvertase (PC) in a post-translational process. Thus, it wasinvestigated whether or not the Arg-Arg sequence or the Lys-Arg sequencethat are the sequence at the cleavage site of the prohormone convertaseare present in the mouse, rat and human NEFA sequence. As a result, itwas found that in common with mouse, rat and human NEFA sequence, theLys-Arg sequence was present at the amino acid positions 107 and 108from the N-terminal and the positions 199 and 200, and the Arg-Argsequence is present at positions 188 and 189 (FIG. 9A). Thus, among thenesfatin sequence, a peptide comprising 82 residues corresponding toamino acid Nos. 25 to 106 was named nesfatin-1 (SEQ ID NO: 15), apeptide comprising 79 residues corresponding to amino acid Nos. 109 to187 was named nesfatin-2 (SEQ ID NO: 16), and a peptide comprising 231residues corresponding to amino acid Nos. 190 to 420 was namednesfatin-3 (SEQ ID NO: 17). In this case, the DNA-binding region (aminoacid Nos. 171-223) which is a known domain structure, is divided intonesfatin-2 and nesfatin-3, the nectin-binding region (amino acid Nos.213-420), the calcium-binding region (amino acid Nos. 254-265 and306-317) and Asp/GLu-rich region (amino acid Nos. 306-317) are containedin the sequence of nesfatin-3, but no known domains are contained in thesequence of nesfatin-1 (FIG. 9B). Therefore, the portion of the sequenceconsisting of the nesfatin-2 and the nesfatin-3 was named nesfatin-2/3(SEQ ID NO: 47). The synthesis of the rat nesfatin-1 peptide (SEQ ID NO:15) and the nesfatin-2 peptide (SEQ ID NO: 16) was referred to YanaiharaInstitute Inc., where they were synthesized by solid-phase synthesis andpurified by reverse phase liquid chromatography. The cDNA prepared fromrat brain was amplified, using primers set forth in SEQ ID NO: 48 andSEQ ID NO: 49, to obtain DNA (SEQ ID NO: 50), and then the DNA was usedto prepare nesfatin-3 peptide (SEQ ID NO: 17), by Post Genome InstituteCo., Ltd. (Shimizu Y. et al.: Nature Biotech 19: 751-755, 2001), by thecell-free protein synthesis.

The PCR primers used:

(SEQ ID NO: 48) Forward Primer: 5′-ATGGAGTATTTAAAAACGCTGAGTGAG-3′ (SEQID NO: 49) Reverse Primer: 5′-TTATGTGTGTGGCTCAAACTTCA-3′

The nesfatin-3 prepared in this manner has a sequence in which Met isadded to the N-terminal (SEQ ID NO: 51).

By the same manner, the cDNA derived from rat brain was amplified, usingprimers set forth in SEQ ID NOs: 52 and 53 to obtain DNA (SEQ ID NO:50), to obtain DNA (SEQ ID NO: 54), and then the DNA was used to preparenesfatin-2/3 (SEQ ID NO: 47), by the cell-free protein synthesis.

The PCR primers used:

(SEQ ID NO: 52) Forward Primer: 5′-ATGGAAGAAGTAGGAAGACTGAGAA-3′ (SEQ IDNO: 53) Reverse Primer: 5′-TTATGTGTGTGGCTCAAACTTCA-3′

Also, in order to prepare antibodies for regions other than those foranti-NAP peptide antibodies prepared in Working Example 3, a sequence inwhich Cys was added to the C-terminal of a sequence corresponding to theamino acid Nos. 24-38 (corresponding to the amino acid Nos. 48-62 of themouse precursor NEFA polypeptide of SEQ ID NO: 5) of NAP-1 peptide(NAP-1Ab: SEQ ID NO: 32), a sequence corresponding to the amino acidNos. 1-9 (corresponding to the amino acid Nos. 190-198 of the mouseprecursor NEFA polypeptide of SEQ ID NO: 5) of NAP-3, and a sequence inwhich Cys was added to the C-terminal of a sequence corresponding to theamino acid Nos. 136-149 (corresponding to the amino acid Nos. 325-338 ofthe mouse precursor NEFA polypeptide of SEQ ID NO: 5) of NAP3 peptide(NAP-1 Ab: SEQ ID NO: 33 and NAP-1 Ab: SEQ ID NO: 34) were prepared.

NAP-1 Ab: (SEQ ID NO: 32)N-ProAspThrGlyLeuTyrTyrAspGluTyrLeuLysGlnValIle Cys-C NAP-C1 Ab: (SEQ IDNO: 33) N-GluTyrLeuLysThrLeuSerGluGluCys-C NAP-C2 Ab: (SEQ ID NO: 22)N-LysGluPheLeuGluProAspSerTrpGluThrLeuAspGlnCys-C

The preparation of each peptide of NAP-1 Ab, NAP-C1 Ab and NAP-C2 Ab wasreferred to ATP Co., Ltd. in which they were synthesized by solid-phasesynthesis and purified by reverse phase liquid chromatography. Eachpeptide of NAP-1 Ab, NAP-C1 Ab and NAP-C2 Ab thus obtained wasconjugated to KLH in a manner similar to that described in WorkingExample 3, and then immunized to rabbits to prepared serum containingantibody to each peptide. From the serum, rabbit IgG was purified usingthe DEAE column, and named NAP-1 IgG (nesfatin-1 IgG), NAP-C1 IgG(nesfatin-C1 IgG), and NAP-C2 IgG (nesfatin C2 IgG).

Each of the prepared nesfatin-1 and nesfatin-3 peptide were subjected toSDS-polyacrylamide gel electrophoresis (12%), and then Western blottingwas carried out for each peptide, by in a manner similar to thatdescribed in Working Example 3, using the nesfatin-1 IgG antibody andthe nesfatin-C2 IgG antibody, respectively.

<Result>

The nesfatin-1 peptide, nesfatin-2 peptide, nesfatin-3 peptide andnesfatin-2/3 peptide that were synthesized by solid-phase synthesis andpurified by reverse phase liquid chromatography were purified by ananalytical C18 reverse phase chromatography to a purity of almost asingle peak. By collecting a portion of the peaks and analyzing withmass spectrometry (nesfatin-1 and nesfatin-2) or SDS-polyacrylamide gelelectrophoresis (12% gel) (nesfatin-3 and nesfatin-2/3), it wasdemonstrated that peptides estimated to be approximately the molecularweight of each peptide of predicted nesfatin-1, nesfatin-2, nesfatin-3and nesfatin-2/3 was synthesized. In rabbits that were immunized withthe conjugate of each peptide of NAP-1 IgG, NAP-C1 IgG and NAP-C2 IgGand KLH, increased in the titer of antibody against these 3 peptideswere observed, and antibody (IgG) against each peptide was obtained.

As the results of the Western blotting with the nesfatin-1 IgG afterelectrophoresis of the nesfatin-1 peptide, a 9.7 kd single band wasobserved, and as the results of the Western blotting with thenesfatin-C2 IgG after electrophoreisis of the nesfatin-3 peptide, a 27.9kd single band was observed (FIG. 9C). Herefrom, it is shown that thenesfatin-1 IgG antibody and the nesfatin-C2 IgG antibody, bond to thenesfatin-1 peptide, the nesfatin-3 (nesfatin-2/3), respectively.

Working Example 11 Immunohistochemical Analysis with NAP-1 IgG andAnti-PC Antibody

In order to demonstrate that nesfatin is processed to producenesfatin-1, the localization of nesfatin and PC was first analyzed by adouble immunohistological stain of the rat hypothalamus with NAP-1 IgG(nesfatin-1 IgG) prepared in Working Example 10 and anti-prohormoneconvertase antibody (anti-PC).

Brain tissue sections were prepared in the following method. Eightweek-old Wistar rats (purchased from CHARLES RIVER LABORATORIES Japan,Inc.) were anesthetized by an intraperitoneal injection of 40 mg/kg ofpentobarbital sodium, and then thoracotomy was performed to inject 50 mlof physiological saline (0.85% NaCl) into the heart, and then PBScontaining 4% paraformaldehyde (20 mM phosphate buffer, 150 mM NaCl, pH7.2) was injected and circulated using a 400 ml peristatic pump toperfusion fix the brain. The brain was then extracted, and the portioncontaining the hypothalamus was excised, and was fixed overnight at 4°C. in PBS containing 4% paraformaldehyde. The fixed brain sample wasimmersed in PBS containing 10% sucrose for 4 hours, PBS containing 15%sucrose for 4 hours, and PBS containing 20% sucrose overnight at 4° C.Then the brain sample was immersed in an OCT compound and frozen in dryice-acetone to prepare the embedded block. From the block prepared,sections of 6 μm in thickness were prepared at −20° C. using a cryostat,and then were air-dried on a silane-coated slide (MAS coat slideprepared by Matsunami). Tissue sections on the slide were treated with3% hydrogen peroxide for 5 minutes, and then washed twice in PBS for 5minutes.

The slide glass having a brain tissue section placed thereon was blockedby treating with 10% goat normal serum (Nichirei Corp., Cat. No. 426042)at room temperature for 10 minutes, and then was reacted to NAP-1 IgG(nesfatin-1 IgG: Working Example 10) diluted 1/500 in PBS containing 5%BSA at room temperature for 1 hour. The slide glass to which antibodywas reacted was washed in PBS three times for 5 minutes, and theHistofine Simple Stain Rat MAX PO (Nichirei Corp., Cat. No. 414181) wasreacted thereto, and after washing in PBS, color was developed using4-chloro-1-naphthol (ICN, Cat. No. 980611: 4-chloro-1-naphtholstabilized chromogen). The slide glass after color development waswashed in PBS three times for 5 minutes, and then immersed in 0.1Mglycine buffer (pH 2.2) for 1.5 hours (said buffer was changed every 30minutes), and washed in PBS three times for 5 minutes. The slide glasswas further blocked with 10% goat normal serum (Nichirei Corp., Cat. No.426042) at room temperature for 10 minutes. The slide glasses that weretreated to this point were divided into two groups: one was reacted toan antibody (PC-1/3: Chemicon, Cat. No. AB1260) that recognizesprohormone convertase subtypes PC1 and PC3, and the other was reacted toantibody (PC-2: Chemicon, Cat. No. AB1262) that recognizes prohormoneconvertase subtype PC2 as an antibody solution diluted in PBS containing0.5% BSA at room temperature for 1 hour. The slide glass was then washedin PBS three times for 5 minutes, and to the slide glass was reacted togoat anti-rabbit IgG-Alexa594 conjugate (Molecular Probes, Cat. No.A11008) diluted 1/200 in PBS containing 0.5% BSA at room temperature for30 minutes. Then the slide glass was washed in PBS three times for 5minutes and slightly air-dried, and then mounted with glycerol and acover slip, and the perimeter was sealed with clear manicure to preparea specimen. The specimen was examined as a phase contrast image of4-chloro-1-naphthol stain by a cofocal laser microscope (Bio-Rad Inc.MRC-1024 cofocal laser scanning equipment+Nikon's Eclipse E800 uprightmicroscope) and fluorescence with Alexa 594 was examined as afluorescent image by an excitation beam with the Krypton-Argon laserusing a 605±30 nm band filter.

<Result>

In the immunohistochemical image for the rat hypothalamus tissue, astained image with nesfatin-1 IgG is shown in an upper panel and a lowerpanel in A of FIG. 10, a fluorescence image with PC-1/3 in the upperpanel in B of FIG. 10, and a fluorescence image with PC-2 in the lowerpanel in B of FIG. 10. The upper panel in A of FIG. 10 and the lowerpanel in B of FIG. 10, and lower panel in A of FIG. 10 and the lowerpanel in B of FIG. 10 represent a color image and a fluorescence imageat the same field. As shown in the upper panel and the lower panel in Aof FIG. 10, cells stained with nesfatin-1 IgG was observed in the neuralcells in the rat brain. Many of cells which were stained withanti-PC-1/3 and anti-PC-2 antibody also corresponded to the nesfatin-1IgG positive cells. This indicated that in cells expressing nesfatinprohormone convertases PC1/3 and PC2 are simultaneously expressedsuggesting the possibility that nesfatin undergoes processing byprohormone convertase.

Working Example 12 Study on the Effect of Intracerebral Administrationof nesfatin-1, nesfatin-2, nesfatin-3 and nesfatin-2/3 on Food IntakeControl

The site of nesfatin of which the activity of food intake suppression ispresent was investigated.

Samples used for intracerebral administration were PBS alone (vehiclegroup), or nesfatin-1, nesfatin-2, nesfatin-3 or nesfatin-2/3 preparedin Working Example 10 each dissolved in PBS, and 25 pmol wasadministered per individual into the third ventricle immediately beforethe dark period. Rats used were Wistar rats (purchased from Nippon SLC)and were housed as in Working Example 6, and among 8-9 week-old maleWistar rats, those individuals weighing 200-250 g were selected and used(N=5). The technique of intraventricular administration and themeasurement of the amount of food intake in rats were carried out in amethod similar to those in Working Example 6. In the measurement of theamount of food intake, the reduced amount of food at 1 hour afteradministration (0-1 hr) and 2 hours after administration (1-3 hrs) wasdetermined as food intake.

In the same condition above, 1, 5 or 25 pmol of nesfatin-1 as a samplewas administered per rat individual into the third ventricle immediatelybefore the dark period. In this experiment, the amount of food intakewas determined at 1 hour after administration (0-1 hr).

<Result>

Compared to the control group (Cont), as shown in A of FIG. 11, theintraventricular administration of nesfatin-2, nesfatin-3 and nesfatin2/3 did not exhibit any significant changes in the amount of food intake(0-1 hr: a-1 in A of FIG. 11; 1-3 hr: a-2 in A of FIG. 11A). On thecontrary, the intra-celebroventricular administration of 25 pmol ofnesfatin-1 exhibits marked effect on the suppression of feeding. Asshown in B of FIG. 11, the intraventricular administration of 1, 5 or 25pmpl of nesfatin-1 per rat individual did exhibit the decreased amountof food intake, with the increased amount of the administerednesfatin-1, compared to the saline group (O). In particular, asignificant decreased amount of food intake was observed in theadministration of 5 pmol or 25 pmpl of nesfatin-1. These resultsrevealed that the activity of suppressing food intake by nesfatin islocalized in nesfatin-1.

Working Example 13 Changes in the Amount of Food Intake and Body Weightin the Intraventricular Continuous Administration of nesfatin-1

Effect of intraventricular continuous administration of nesfatin-1 onchanges in the amount of food intake and body weight was investigated.

The administration of nesfatin-1 was carried out into the ventricle byan osmotic pump for 10 consecutive days. Among 8 week-old male Wistarrats (purchased from Nippon SLC), those individuals weighing 200-250 gwere used. The rats received intraperitoneal administration of 40 mg/kgbody weight of pentobarbital sodium for anesthesia. After the hair wasremoved from the head, they were fixed in a brain stereotaxic apparatus(David Kopf Instruments, Model 962) and the scalp was incised and a 23Gneedle (guide cannula) was implanted so that the tip reaches the thirdventricle (2.5 mm behind the bregma, 9.5 mm from the surface). One weekafter implanting the guide cannula, the animals were subjected to theexperiment. The osmotic pump used was Alzet's Model 2002. Nesfatin-1(nesfatin-1 administration group) dissolved in physiological saline thatwas filter-sterilized with a 0.22 μm Millex GV filter (Millpore Co.Ltd.), or sterilized physiological saline alone (control group) wereinjected into the osmotic pump immediately before use, and were primedin sterilized physiological saline from the day before use. The osmoticpump to which each sample was connected to a tube connected to theinjection cannula, and through the guide cannula previously implantedthe injection cannula was fixed so that the tip of the cannula reachesthe third ventricle of the rat. In this form of use, the sample isinjected to the third ventricle at a flow rate of 12 μl per 24 hours,and in the nesfatin-1 administration group, it is equivalent to 5 pmolof nesfatin-1 per day administered to the ventricle. After confirmingthe rats into which the osmotic pump was implanted recovered fromanesthesia, they were placed in individual cages where they were keptunder the condition that they can take the powder chew and water atlibitum. The measurement of the amount of food intake started from theday after the cannula and the osmotic pump were implanted, and theweight of the residual food was measured at 9 a.m. every day, and bydetermining the difference from the weight of the food on the previousday, the amount of food intake of the day (24 hours) was calculated.From day 6 after the start of administration, body weight was measuredat the time of measuring the amount of food intake, and changes in bodyweight were also recorded.

<Result>

As shown in A of FIG. 12, in the nesfatin-1 administration group, adecrease in the amount of food intake was noted from day 1 after thestart of administration, and the decrease was maintained during theperiod of measurement as compared to the control group. Also as shown inB of FIG. 12, the rate of increase in body weight in the nesfatin-1administration group has significantly declined as compared to thecontrol group.

Working Example 14 Study on Food Intake Control by the IntraventricularAdministration of Anti-nesfatin-1 Antibody or Anti-nesfatin-3 Antibody

In Working Examples 12 and 13, the effect of decreasing the amount offood intake by the intraventricular administration of nesfatin-1 peptidewas found. In order to further confirm this fact, NAP-1 IgG (nesfatin-1IgG) that recognizes nesfatin-1, NAP-C1 IgG (nesfatin-C1 IgG), or NAP-C2IgG (nesfatin-C2 IgG) that recognizes nesfatin-3 were intraventricularlyadministered to rat to study its effect on the amount of food intake. Tostudy the relationship between the nesfatin cleaved by prohormoneconvertase to generate nesfatin-1, and the food intake control, a mutant(KR-AA mutant: Mut) in which a Lys-Arg sequence corresponding to aminoacid numbers 84 and 85, among a sequence set forth in SEQ ID NO: 26, isreplaced with a Ala-Ala sequence was prepared and investigated effect onthe intraventricular administration in rats.

Samples used for intraventricular administration were IgG (control IgGgroup) purified from unimmunized rabbits with PBS, NAP-1 IgG (nesfatin-1IgG), NAP-C1 IgG (nesfatin-C1 IgG) or NAP-C2 IgG (nesfatin-C2 IgG)prepared in Working Example 11, which was dissolved in PBS, and 5 μl (5μg) of them was administered to the third ventricle. The rats used wereWistar rats (purchased from Nippon SLC) and were housed as in WorkingExample 6, and among 8-9 week-old male Wistar rats, those individualsweighing 200-250 g were selected and used. The technique ofintraventricular administration and the measurement of the amount offood intake were carried out in a method similar to those in WorkingExample 7. In the measurement of the amount of food intake, the reducedamount of food at 3 hours after administration (0-3 hr) and 3 hours over3 hours after administration (3-6 hrs) was determined as food intake.

The preparation of the KA-AA mutant was carried out by altering anesfatin gene from the mature mouse prepared in Working Example 5 and byexpressing the gene. Two fragments were prepared using the plasmide inwhich the nesfatin gene from the mature mouse obtained in WorkingExample 5 was cloned to pGEM-11Zf(+), by PCR method. The first fragmentwas prepared using 5 ng of the plasmid and by carrying out PCR reactionwith a set of mNucB2-F360-[Sac2Thr] primer and mNucB2-[KR-AA] R583primer.

mNucB2-F360 [Sac2Thr]: (SEQ ID NO: 56)5′-GGTTCCGCGGGTCTGGTTCCGCGTGGTTCTGTTCCTATCGATGTGGA CAAGACCAA-3′ mNucB2[KR-AA] R583: (SEQ ID NO: 57) 5′-CTTCTTGAGCAGCCAGCTCATCCAGTCTCGTCCTCA-3′

The PCR reaction comprised a denaturation reaction at 90° C. for 1minute, followed by 20 cycles of reaction comprising denaturation at 98°C. for 10 seconds, annealing at 60° C. for 30 seconds, and extension at68° C. for 1 minute, to prepare the amplified DNA (Stratagene'sPfuTurbo® polymerase, Cat. No. 600250 was used).

The second fragment was prepared using 5 ng of DNA fragment of theplasmid and by carrying out PCR reaction with a set of mNucB2[KR-AA]F612 primer and mNucB2-R1527[NotI] primer.

mNucB2 [KR-AA] F612: (SEQ ID NO: 58)5′-GAGCTGGCTGCTCAAGAAGTAGGAAGACTGCGGGATGCT-3′ mNucB2-R1527 [NotI : (SEQID NO: 59) 5′-GGTTGCGGCCGCACTTTATGTGTGTGGCTCAAAC-3′

The PCR condition is the same as the amplification condition for thefirst DNA fragment.

To prepare 2 DNA fragments, the samples obtained by two PCR reactionswere added to a reaction mixture so that a concentration of the reactionmixture is 5 μl each per 50 μl. Using 0.25 μM each of His-Thr-For[SpeI]primer and the above mNucB2-R1527-[NotI] primer, and a reaction mixtureof 1× concentrated Pfu buffer, 2.5 units of PfuTurbo DNA polymerase(stated above: Strategene) and 0.2 mM of dNTPs (Promega: C1141), adenaturation reaction was carried out at 90° C. for 1 minute, followedby 20 cycles of reaction comprising denaturation at 98° C. for 10seconds, annealing at 60° C. for 30 seconds, and extension at 68° C. for1 minute, to prepare the amplified DNA.

His-Thr-For[SpeI]: (SEQ ID NO: 60)5′-GGTTACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTC TGGTTCCGCGT-3′.

The PCR product obtained was subjected to 1% agarose gel electrophoresisto excise an amplified band, which was purified with QIAEX-II kit(QIAGEN). The amplified DNA excised was cleaved with restriction enzymesSpeI and NotI, and ligated to pET41a(+) plasmid vector (Novagen) cleavedwith restriction enzymes SpeI and NotI, using the Quick DNA ligase kit(New England Biolabs). From the cloned DNA, a clone, which has no errorsin mutant portions introduced after analyzing DNA sequences in a similarmanner to Working Example 5 and other nucleotide sequences, can beselected to obtain a vector for expressing the KR-AA mutant. Theexpression and purification were carried out in a similar manner toWorking Example 5. The amino acid sequence of the KR-AA mutant obtainedis set forth in SEQ ID NO: 61. Wt (normal nesfatin), which was preparedin Working Example 5, was used. 5 pmol each of the KR-AA mutant (Mut)peptide and the normal nesfatin (Wt) peptide were administered to thethird ventricle of rats in a similar manner to Working Example 6. One,three, six and 12 hours thereafter, the weight of the food wasdetermined to record the amount of food intake, which was used as anindex of feeding behavior.

<Result>

In each of the administration into the third intraventricularadministration group of control IgG, NAP-1 IgG (nesfatin-1 IgG), NAP-C1IgG (nesfatin-C1 IgG) or NAP-C2 IgG (nesfatin-2 IgG), the amount of foodintake during 0-1 hour after the administration is graphically shown ina-1 of FIG. 13A, the amount of food intake during 3-6 hours isgraphically shown in a-2 of FIG. 13A, and the amount of food intakeduring 6-9 hours is graphically shown in a-3 of FIG. 13A. In 0-3 hour(a-1 of FIG. 13A) and 3-6 hours (a-2 of FIG. 13A), as compared to thecontrol IgG administration rats, individuals that received NAP-1 IgG(nesfatin-1 IgG) exhibited a significant increase in the amount of foodintake (P<0.01 for 0-3 hour, P<0.05 for the 3-6 hours), but in thegroups that received NAP-C1 IgG (nesfatin-C1 IgG) or NAP-C2 IgG(nesfatin-C2 IgG), no significant difference was noted as compared tothe vehicle group. In the results for 6-9 hours, no significantdifference was noted in each of the control IgG group, the NAP-1 IgG(nesfatin-1 IgG) group, the NAP-C1 IgG (nesfatin-C1 IgG) group andNAP-C2 IgG (nesfatin-C2 IgG) group. This result indicated that thefunction of endogenous nesfatin-1 is inhibited by antibody againstnesfatin-1, resulting in the increase of amount of food intake. Thisdemonstrated that a substance that inhibits the function of nesfatin-1has an effect of enhancing the amount of food intake, in addition to theeffect of suppressing food intake by the intraventricular administrationof the nesfatin-1 polypeptide as demonstrated in Working Example 13.

Further, the results of an effect on the amount of food intake in rats,in which 5 pmol each of a recombinant mouse nesfatin (Wt) and the mutant(KR-AA mutant: Mut) in which a Lys-Arg sequence corresponding to aminoacid numbers 84 and 85 was replaced with an Ala-Ala sequence, wasadministered to intraventricular in the rats, are shown in FIG. 13B. InFIG. 13B, the amount of food intake at 0-1 hour, 1-3 hour and 3-6 hoursafter the administration were graphically shown in b-1, b-2, b-3,respectively. As shown in b-1 to b-3, in the rats in which 5 pmol of Wt(normal nesfatin) was administered, a significant control activity offood intake was observed at each of the period, while no controlactivity of food intake was observed in the nesfatin (Mut) in which amutation was introduced into the site cleaved by prohormone convertase.Herefrom, it was demonstrated that the step of nesfatin-1 processed byprohormone convertase is important for nesfatin to functionate.

Working Example 15 Study on the Effect of Continuous IntraventricularAdministration of Antisense RNA Against the NEFA Gene on Food Intake andBody Weight

In order to further investigate the relationship between the expressionof the nesfatin (NEFA) gene and the control of food intake and bodyweight, antisense RNA that suppresses the expression of nesfatin genewas continuously administered into the third ventricle and its effectwas investigated.

As the antisense RNA against nesfatin-1 gene, was used a morpholino RNAwhich sandwiches a translational start site, as follows.

Nesfatin-1 antisense RNA: 5-ATGGTCCTCCACCTCATCTTCAGAG-3 (SEQ ID NO: 31)

The administration of nesfatin antisense RNA was carried out into theventricle by an osmotic pump for 12 consecutive days using an osmoticpump. Among 8 week-old male Wistar rats (purchased from Nippon SLC),those individuals weighing 200-250 g were selected and used. The ratsreceived intraperitoneal administration of 40 mg/kg body weight ofpentobarbital sodium for anesthesia. After the hair was removed from thehead, they were fixed in a brain stereotaxic apparatus (David KopfInstruments, Model 962) and the scalp was incised and a 23G needle(guide cannula) was implanted so that the tip reaches the thirdventricle (2.5 mm behind the bregma, 9.5 mm from the surface). One weekafter implanting the guide cannula, the animals were subjected to theexperiment. The osmotic pump used was Alzet's Model 2002 that was primedin sterilized physiological saline from the day before use. Nesfatinantisense RNA (antisense administration group) dissolved inphysiological saline that was filter-sterilized with 0.22 μm Millex GVfilter, or missence RNA (ATcGTgCTCCACgTCATCTaCAcAG) sterilized in thesame manner that dissolved in sterilized physiological saline (controlgroup) were injected into the osmotic pump immediately before use. Theosmotic pump to which each sample was connected to a tube connected tothe injection cannula, and through the guide cannula previouslyimplanted the injection cannula was fixed so that the tip of the cannulareaches the third ventricle of the rat. In this form of use, the sampleis injected to the third ventricle at a flow rate of 12 μl per 24 hours,and 40 μg each of antisense RNA and missense RNA were injected. Afterconfirming the rats into which the osmotic pump was implanted recoveredfrom anesthesia, they were placed in individual cages where they werekept under the condition that they can take the powder food and water atlibitum. The measurement of the amount of food intake started from theday after the cannula and the osmotic pump were implanted, and theweight of the residual food was measured at 9 a.m. every day and, bydetermining the difference from the weight of the food on the previousday, the amount of food intake of the day (24 hours) was calculated.From day 6 after the start of administration, body weight was measuredat the time of measuring the amount of food intake, and changes in bodyweight were also recorded.

On day 12 after the start of administration, the individuals in eachgroup were sacrificed, and the hypothalamus in the brain was removed.Using the sample extracted, the expression of nesfatin was confirmed byWestern blotting with nesfatin-1 Ab. Western blotting was carried out ina manner similar to those described in Working Examples 13 and 12. Theconcentration of bands stained in Western blotting was determined bydensitometry.

<Result>

Changes in the amount of food intake during 24 hours for the controlgroup and the antisense administration group to which nesfatin antisenseRNA was administered are shown in A of FIG. 14 and changes in bodyweight are shown in B of FIG. 14. As compared to the control group, anincrease in the amount of food intake (A of FIG. 14) was noted in theantisense administration group from the day 1 after the start ofadministration, and the amount of food intake was always greater thanthe control group during the measurement period (until day 12 after thestart of administration). For changes in body weight (B of FIG. 14), nodifference was noted on day 6 after the start of administration betweenthe control group and the antisense administration group, but on day 7and after a significant increase in body weight (P<0.05) was noted inthe antisense administration group as compared to the control group, andthe difference expanded from day 9 to day 11 of the measurement period.In the Western blotting analysis of nesfatin-1 using the rathypothalamus sacrificed on day 12, the expression of nesfatin-1 wassignificantly decreased in the antisense administration group (the bandintensity by densitometry was 8.5±0.7 AU) compared to the control group(the band intensity indicated 14.3±1.2 AU, which was determined usingdensitometry). From the result, it was revealed that theintraventricular administration of nesfatin antisense RNA has an effectof suppressing the expression of nesfatin-1 and of enhancing the amountof food intake and body weight gain.

Working Example 16 Production of Recombinant nesfatin-1 by a Recombinant

In order to prepare nesfatin-1 in large quantities, a method ofproducing recombinant nesfatin-1 using a recombinant was investigated.

A gene encoding mouse nesfatin-1 was obtained, and an expression vectorwas constructed by binding the gene of glutathione S-transferase (GST)and histidine tag to the N-terminal of the nesfatin-1 gene, so that acleavage site (-Leu-Val-Pro-Arg-Gly-Ser-) cleaved using thrombinmediates the amino acid sequence of histidine tag and the amino acidsequence of mouse nesfatin-1 in the protein after translation. The geneof mouse nesfatin-1 was obtained using mouse brain cDNA (Clontech) bytwo runs of PCR (nested PCR). The first PCR used a forward primer(mNucB2-F337: SEQ ID NO: 35) and a reverse primer (mNucB2-R712: SEQ IDNO: 36) at 100 μM each, Pyrobest DNA polymerase (Takarabio K.K. R005A),the reaction buffer attached and dNTP and was carried out according tothe attached protocol. The PCR reaction comprised, after reaction of 90°C. for 1 minute, a temperature condition of 30 cycles of 98° C. for 10seconds and 68° C. for 1 minute, and then a reaction at 68° C. for 2minutes.

Forward primer (mNucB2-F337): 5′-GCACGCTGAC CGCTCTGGAAG-3′ (SEQ ID NO:35) Reverse primer (mNucB2-R712): 5′-CAAATGTGTT AGGATTCTGGTGGTTCA-3′(SEQ ID NO: 36)

Using 0.5 μl of the PCR product obtained, the second run of PCR wascarried out using a forward primer (mNucB2-N3-[SacI-Thr]) and a reverseprimer (mNucB2-R389-[NotI]) at 100 μM each, and using Pyrobest DNApolymerase as in the first PCR. The PCR reaction comprised, afterreaction of 90° C. for 1 minute, a temperature condition of 20 cycles of98° C. for 10 seconds, 60° C. for 30 seconds and 68° C. for 1 minute,and then a reaction at 68° C. for 2 minutes.

Forward primer (mNucB2-N3 [SacI-Thr]): (SEQ ID NO: 37)5′-GGTTCCGCGGGTCTGGTTCCGCGTGG TTCTCCTATCGATGTGGACA AGACCAA-3′ Reverseprimer (mNucB2-R589[NotI]): (SEQ ID NO: 38) 5′-GGTTGCGGCCGCTTACCTCTTCAGCTCA TCCAGTCTCG-3′

PCR reaction samples that were subjected to two runs of PCR werepurified with phenol/chloroform extraction, cleaved with restrictionenzymes SacII and NotI, and then subjected to agarose gelelectrophoresis to excise a band corresponding to a length of about 300bp, which was purified with QIAEX-II kit (QIAGEN). The purified PCRproduct of about 300 bp was ligated to pET41a(+) plasmid vector(Novagen) cleaved with restriction enzymes SacII and NotI using theQuick DNA ligase kit (New England Biolabs). The ligated vector wasintroduced into an E. coli strain J409 and the 8 transformants thusobtained were subjected to plasmid extraction on a small scale. Theplasmids thus obtained were analyzed for the nucleotide sequence of thesequence of the nesfatin-1 gene integrated, using the BigDye TerminatorCycle Sequencing FS Ready Reaction kit and ABI377 type DNA sequencer(Perkin-Elmer Inc.). As a result, an expression vector having a gene inwhich the correct sequence of nesfatin-1 has been integrated wasobtained, and this was named pET41a(+)GST-His-LVPRGS-mNAP1.

By introducing the pET41a(+)GST-His-LVPRGS-mNAP1 into E. coli BL21 (DE3)Codon Plus RIPL and expressing, a fusion protein (GST-His-LVPRGS-mNAP1)of GST-Histidine tag-thrombin cleavage sequence-nesfatin-1 wasexpressed. Clones obtained by introducing thepET41a(+)GST-His-LVPRGS-mNAP1 into E. coli BL21 (DE3) Codon Plus RIPLand selecting in a LB medium containing kanamycin was cultured in 10 mlof the LB medium containing kanamycin at 37° C. Culturing was stoppedwhen the absorbance at a wavelength of 600 nm became 0.8. 3 ml of theculture liquid was inoculated into 100 ml of the LB medium containingkanamycin, which was cultured at 37° C. When the absorbance at awavelength of 600 nm became 0.8, 1 ml of 100 mM IPTG was added to inducethe expression of protein. After adding IPTG, a shaking culture wascarried out at 37° C. for 3 hours. The culture liquid was centrifuged at8000 rpm for 20 minutes (4° C.) to collect the cells of E. coli.

From the cells of E. coli thus obtained, the GST-His-LVPRGS-mNAP1 fusionprotein was extracted, and purified with a nickel chelate column (Ni-NTAagarose). The cells were suspended in 20 ml of the Sonication buffer (50mM KH2PO4, 50 mM NaCl, 2 mM DTT, pH 7.5) containing one-foldconcentration of Complete-EDTA free (Roche Diagnostics K.K.) and0.5-fold concentration of BugBuster (Merck Ltd., Novagen, Cat. No.70584), and were disrupted by sonication in ice water for 10 minutes.The sonicated sample was centrifuged at 15,000 rpm for 20 minutes, andthe supernatant was collected. 10 ml of the supernatant obtained wasapplied to 1 ml Ni-NTA agarose column equilibrated with the Lysis buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and washed twicein 10 ml of the Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mMimidazole, pH 8.0). The column after washing was eluted twice with 2.5ml of the Elution buffer (50 mM Na2HPO4, 300 mM NaCl, 250 mM imidazole,pH 8.0), and a fraction containing the eluted GST-His-LVPRGS-mNAP1fusion protein was collected. The extraction supernatant from theresidual cells was similarly treated, and a fraction containingGST-His-LVPRGS-mNAP1 fusion protein was collected.

From the GST-His-LVPRGS-mNAP1 fusion protein, the portion of GST andhistidine tag was removed, and further purification,GST-His-LVPRGS-mNAP1 fusion protein bound to the GST resin was subjectedto thrombin treatment and to purification with reverse phasechromatography in order to remove E. coli-derived lipopolysaccharide(LPS) that may act as an inflammatory substance. The buffers at thisstage and after were those confirmed to be LPS-free. 7.2 ml of thefraction containing the GST-His-LVPRGS-mNAP1 fusion protein was washedone-fold concentration of GST Bind/Wash buffer (Merck Ltd., Novagen,Cat. No. 70571), which was finally added to the GST resin (Merck Ltd.,Novagen, Cat. No. 70541) (equivalent to 7.2 ml) suspended in 3 ml of theGST Bind/Wash buffer, and gently stirred at 20° C. for 1 hour. Aftercollecting the resin by centrifugation, said resin was washed twice in36 ml of the GST Bind/Wash buffer. To the washed resin, 3.6 ml of asolution of 20 units/ml of thrombin in PBS was added and suspended, andreacted at 20° C. for 20 hours under gentle stirring. The resin afterthe reaction was delivered in 1.8 ml in a filter-attached cup(Millipore) with a pore size of 0.22 μm, centrifuged at 3,000 rpm for 2minutes, and the filtered thrombin-treated sample was collected. To 450μl of the thrombin-treated sample was added 50 μl of acetic acid toprepare a sample for C18 reverse phase chromatography. The reverse phasechromatography comprised the elution with a gradient of acetonitrile in0.1% trifluoroacetic acid, and the gradient was set at 10%acetonitrile:10 minutes/10-20% acetonitrile gradient:60 minutes/30-40%acetonitrile gradient:40 minutes/40-60% acetonitrile gradient:5 minutes.Protein eluted from the column was monitored by measuring absorbance ata wavelength of 280 nm. By examining the fractions eluted withacetonitrile gradient by SDS-PAGE and Western blotting, nesfatin-1 wasfound to be eluted at an acetonitrile concentration of 36.2%. Thus, thisfraction was collected, lyophilized, and dissolved again in distilledwater for injection, which was used to determine protein concentrationby absorbance and LPS content by Endospacy assay (Seikagaku Kogyo).

<Result>

From 100 ml of the culture liquid, about 7 mg of crudeGST-His-LVPRGS-mNAP1 was purified with Ni-NTA-agarose, and the recoveryof nesfatin-1 that was thrombin-treated or highly purified by C18reverse phase chromatography was 472.5 μg. The amount of LPS containedin the highly purified nesfatin-1 was about 4 μg relative to 1 μg ofnesfatin-1. Furthermore, when the highly purified nesfatin-1 wasintraventricularly administered to rats in a method similar to that inWorking Example 13, the effect of suppressing food intake and/orsuppressing body weight gain was noted. This indicated that theproduction of active nesfatin-1 is possible by expression andpurification using a recombinant.

Working Example 17 Study on the Effect of nesfatin-1 Administration intothe Third Ventricle of Zucker fa/fa Rats on Food Intake Control

As described in the conventional technology, many of obese people orpatients with adiposis exhibit resistance to leptin, which poses aproblem in the pathology and treatment. Thus, using Zucker fa/fa rats(Michael et al., Nature Genetics, Vol. 13, pp. 18-19, 1996), an animalpathological model of leptin-resistance, the effect of nesfatin-1 on thecontrol of the amount of food intake was investigated.

As rats, 8 week-old male Zucker fa/fa (Zucker) rats and Zucker +/+(Lean)rats as the control animal were purchased from Nippon Charles River, andwere housed in a cycle of 12 hours of the light period from 6 a.m. to 6p.m. and 12 hours of the dark period from 6 p.m. to 6 a.m. the nextmorning and fed a powder food (Nippon Clea, CE-2), and kept at 22° C.,and throughout the experiment period, a similar condition was continuedin housing. After preliminary housing of the purchased rats for over oneweek, individuals weighing 200-250 g were selected from among 9 to 10week-old individuals.

The sample used for administration was one in which the recombinantmouse nesfatin-1 prepared in Working Example 16 was dissolved to 5 pmolin 5 μl of PBS, and as the control sample physiological saline (Saline)was used. Five μl of the samples prepared were administered into thethird ventricle of each rat for the group of five each of Zucker ratsand the Lean rats per group (the Zucker/nesfatin-1 group, theLean/nesfatin-1 group, the Zucker/Saline group, the Lean/Saline group).The timing of administration was immediately before beginning the darkperiod when food intake behavior is enhanced, and the method ofadministration was similar to that described in Working Example 6.

After intraventricular administration, the amount of food intake wasdetermined by measuring the amount decreased of the powder food for eachrat during 0-1 hour, 1-3 hours and 3-6 hours. For test of significantdifference, analysis of variance was used.

<Result>

A of FIG. 15 shows the result measured of food intake for the group (theLean/nesfatin-1 group) in which nesfatin-1 was administered and for thegroup (the Lean/Saline group) in which physiological saline wasadministered to the Lean rats, and B of FIG. 15 shows the resultmeasured of food intake for the group (the Zucker/nesfatin-1 group) inwhich nesfatin-1 was administered and the group (the Zucker/Salinegroup) in which physiological saline was administered to the Zuckerrats. In a result (A of FIG. 15) for the control animal, Lean rats, theamount of food intake during 0-1 hour and during 1-3 hours was decreasedin the nesfatin-1 administration group relative to the Salineadministration group (P<0.001). No difference was noted in the amount offood intake during 3-6 hours. In Zucker, a leptin-resistant animal, asin Lean, a significant reduction in the amount of food intake during 0-1hour and during 1-3 hours was noted in the nesfatin-1 administrationgroup relative to the Saline administration group (P<0.001). During 3-6hours, a significant reduction (P<0.05) was also noted (B in FIG. 15).The above suggests that the effect of suppressing food intake bynesfatin-1 was exhibited without the effect of leptin and it isconsidered effective under a leptin-resistant condition.

Working Example 18

Study on the Effect of Intraperitoneal Administration of nesfatin-1 onthe Control of the Amount of Food Intake in Mice

It was demonstrated in the experiment of intraventricular administrationin rats that nesfatin and nesfatin-1 are involved in controlling theamount of food intake. In order to study the effect on other animalspecies, the administration experiment on mice was carried out.Considering practical utility as a pharmaceutical, it was thought to beimportant that the peripheral administration is also effective, and thusan intraperitoneal administration was selected as an administrationroute. Furthermore, an administration experiment was also carried out onthe Agouti-yellow (c57BL/6J-A^(y)/a) mice which are animal model ofobesity in which the function of suppressing food intake by MC3R/MC4Rhas been inhibited by the excessive expression of the Agouti protein.

The experimental animal was 7 week-old male ICR mice purchased fromJapan SLC, Inc., and were housed after purchase in a cycle of 12 hoursof the light period from 6 a.m. to 6 p.m. and 12 hours of the darkperiod from 6 p.m. to 6 a.m. the next morning with free access to apellet food (Nippon Clea, CE-2), kept at 22° C., and housing wascontinued in a similar condition during the experiment period. The micepurchased were subjected to preliminary housing for over one week,individuals weighing 35-40 g were selected among 8 to 9 week-oldindividuals and were used in the experiment.

The sample used in the administration was the recombinant mousenesfatin-1 prepared in Working Example 16 dissolved in 200 μl ofphysiological saline so as to contain 2 nmol, 10 nmol or 50 nmol, and asthe control sample, physiological saline (Saline) alone was used. Usinga tuberculin syringe equipped with a 25G needle, 200 μl each of thesample was administered once into the abdominal cavity of each mouse (5animals per group), and the time of administration was immediatelybefore the start (6 p.m.) of the dark period.

In the experiment on the animal model of obesity, C57BL/6J mice wereused as the control group and Agouti-yellow mice were used as the animalmodel of obesity, and these animals were purchased from Nippon CharlesRiver (Jackson Lab.). The housing condition and the week age of the miceused were similar to those for the ICR mice, whereas individualsweighing 25-28 g for the control group mice and individuals weighing31-38 g for the Agouti-yellow mice were selected and used. The sampleused in the administration was recombinant mouse nesfatin-1 dissolved in200 μl of physiological saline so as to contain 10 nmol, and as thecontrol sample, physiological saline (Saline) alone was used (16 animalsper group). Other conditions were similar to those in the above.

Each mouse that received administration was placed in an individualcage, and during 0-3 hours after the administration the weight decreasedof the pellet food was measured to determine the amount of food intake.For testing of significant difference, analysis of variance was used.

<Result>

The result measured of the amount of food intake during 0-3 hours afteradministration when nesfatin-1 or physiological saline wasintraperitoneally administered into ICR mice is shown in A of FIG. 16.In the result of A of FIG. 16, a decreases in the amount of food intakewas noted in the mice that received 2 nmol, 10 nmol and 50 nmol ofnesfatin-1 per mouse relative to the control (Saline group), andstatistically significant decreases in the amount of food intake werenoted in 10 nmol (p<0.05) and 50 nmol (p<0.005). This indicated thatsince nesfatin-1 exhibits an activity of suppressing food intake in miceas well as in rats, it has an effect of controlling food intake in manyspecies, and administration from not only via the brain but via theperiphery such as the abdominal cavity is effective in suppressing foodintake. Also, it was demonstrated that the intraperitoneallyadministered nesfatin-1 is has an effect of suppressing food intake inthe early stage after administration.

Also, in the experiment on the mouse model of obesity, the resultmeasured of the amount of food intake when physiological saline (Cont)or nesfatin-1 (10 pmol) was administered to the mice (c57BL/6J) of thecontrol group is shown in B of FIG. 16, and the result measured of theamount of food intake when physiological saline (Cont) or nesfatin-1 (10pmol) was administered to the Agouti-yellow mice, the mouse model ofobesity, is shown in C of FIG. 16. As a result, both in the mice of thecontrol group and the mice of the Agouti-yellow mice, the amount of foodintake was significantly decreased in the mice that received theintraperitoneal administration of nesfatin-1 relative to the control.Since Agouti-yellow mice is a model of obesity in which the function ofsuppressing food intake by melanocortin, a ligand of MC3R/MC4R, does notwork due to the over expression of the Agouti protein, it was suggested,a pharmacological activity was demonstrated in a leptin-resistant modelin the result for the Zucker (fa/fa) rats in Working Example 17, andsimilarly food intake was controlled by a mechanism independent of thatof food intake suppression in the existing Agouti/melanocortin system.

Working Example 19 Study on the Effect of the Intraperitoneal andSubcutaneous Administration Of nesfatin-1 On Controlling the Amount ofFood Intake in Mice

In Working Example 18, it was disclosed that the intraperitonealadministration of nesfatin-1 into mice is also effective in suppressingfood intake, and thus as an example of another peripheraladministration, the effect of subcutaneous administration of nesfatin-1was also examined.

The sample used in the administration was the recombinant mousenesfatin-1 prepared in Working Example 16 dissolved in 200 μl ofphysiological saline so as to contain 10 nmol, and as the controlsample, physiological saline (Saline) alone was used. Using a tuberculinsyringe equipped with a 25G needle, 200 μl each of the samples wasadministered once into the abdominal cavity or the hypodermis of theback of each mouse, and the time of administration was immediatelybefore the start (6 p.m.) of the dark period. Other conditions weresimilar to those in Working Example 18.

<Result>

The result measured of the amount of food intake during 0-3 hours afteradministration when nesfatin-1 (10 nmol) or physiological saline (0) wasintraperitoneally (ip) or subcutaneously (sc) administered into the miceis shown in A of FIG. 17, and that during 0-14 hours is shown in B ofFIG. 17. In A of FIG. 17, the amount of food intake tended to decreasein the groups in which 10 nmol of nesfatin-1 was intraperitoneally (ip)and subcutaneously (sc) administered relative to the physiologicalsaline-administration group (O), and specifically for theintraperitoneal administration, a statistically significant decrease(P<0.05) was noted. In B of FIG. 17 as well, the amount of food intaketended to decrease in the group in which 10 nmol of nesfatin-1 wasintraperitoneally (ip) and subcutaneously (sc) administered relative tothe physiological saline-administration group (O), but in theintraperitoneal administration group (ip) the decrease in the amount offood intake was not significant and in the subcutaneous administrationgroup (sc) the decrease was significant (P<0.005) relative to thephysiological saline-administration group. In the results of both A andB of FIG. 17, the intraperitoneal administration of nesfatin-1 tended toexhibit the effect of suppressing food intake early and the effect bythe subcutaneous administration tended to lag behind. The above suggeststhat for the peripheral administration, nesfatin-1, whetherintraperitoneally or subcutaneously administered, is effective insuppressing food intake. For drugs that act in the brain, the presenceof effect by the peripheral administration is important in practicaluse, and in this regard nesfatin-1 was shown to be useful as apharmaceutical based on the results of Working Examples 18 and 19.

Working Example 20 Study on the Effect of the IntraperitonealAdministration of a Partial Peptide (nesfatin-1n23, nesfatin-1M30,nesfatin-1C29) of nesfatin-1 on the Control of Food Intake in Mice

In Working Examples 10 and 12, nesfatin-1 was found from the cleavagesites in nesfatin of prohormone convertase. In the analyzing thefunction of nesfatin-1, however, it is important to identify thefunctional site of said peptide, and also in its application intopharmaceuticals, the short amino acid length of the peptide isconsidered advantageous in terms of production, dosage, antigenicity andthe like. Thus, in order to examine in further detail the sites havingan activity of suppressing food intake, partial peptides were preparedfrom the structure of nesfatin-1 comprising a 82-amino acid length, andan experiment was carried out to measure the amount of food intake whenthey were intraperitoneally administered to mice.

Of the amino acid sequence (SEQ ID NO: 14) of mouse nesfatin-1 derivedfrom the sequence of mouse nesfatin, a sequence of amino acid numbers 1to 23 from the amino terminal was termed as nesfatin-1N23, that of aminoacid numbers 23 to 53 as nesfatin-1M30, and that of amino acid numbers54 to 82 as nesfatin-1C29.

nesfatin-1N23: (SEQ ID NO: 42)ValProIleAspValAspLysThrLysValHisAsnThrGluProVal GluAsnAlaArgIleGluPronesfatin-1M30: (SEQ ID NO: 41)ProAspThrGlyLeuTyrTyrAspGluTyrLeuLysGlnValIleGluValLeuGluThrAspProHisPheArgGluLysLeuGlnLys nesfatin-1C29: (SEQ ID NO:43) AlaAspIleGluGluIleArgSerGlyArgLeuSerGlnGluLeuAspLeuValSerHisLysValArgThrArgLeuAspGluLeu

Each peptide of nesfatin-1N23, nesfatin-1M30 and nesfatin-1C29 used wasa synthetic peptide of which production was referred to Biologica Co.,Ltd. and was purified by HPLC to a purity of 95% or higher. Each peptidewas prepared in physiological saline so as to contain 50 nmol per 200μl, which was used as the sample, and as the control samplephysiological saline (Vehicle) alone was used. Using a tuberculinsyringe equipped with a 25G needle, 200 μl each of the samples wasadministered once into the abdominal cavity of each mouse (5 animals pergroup), and the time of administration was immediately before the start(6 p.m.) of the dark period. Mice used were male ICR mice (Nippon SLC)and the housing condition was similar to those in Working Example 18.

Each mouse that received administration was placed in an individualcage, and during 0-3 hours after the administration the weight decreasedof the pellet food was measured to determine the amount of food intake.For testing of significant difference, analysis of variance was used.

Comparison by the alignment of the amino acid sequences of human, ratand mouse nesfatin-1 was also carried out. Using the amino acid sequenceof human nesfatin-1 (SEQ ID NO: 13), mouse nesfatin-1 (SEQ ID NO: 14)and rat nesfatin-1 (SEQ ID NO: 15), alignment was carried out using theCLUSTAL-W method (Higgins et al., Nucleic Acids Research, Vol. 22, pp.4673-4680, 1994).

<Result>

The result measured of the amount of food intake during 0-3 hours afteradministration when physiological saline (Vehicle), nesfatin-1N23(-N23), nesfatin-1M30 (-M30) and nesfatin-1C29 (-C29) wasintraperitoneally administered into mice is shown in A of FIG. 18. Inthe nesfatin-1M30-administration group (N-1b) relative to the controlgroup (Vehicle) that received physiological saline, a significantdecrease (P<0.02) in the amount of food intake was noted. However, inthe nesfatin-1N23-administration group (N-1a) and thenesfatin-1C29-administration group (N-1c), no significant decrease orenhancement in the amount of food intake was noted. The above indicatedthat nesfatin-1M30 is the most important functional site for theactivity of nesfatin-1 (and nesfatin) of suppressing food intake. Also,since the intraperitoneal administration of nesfatin-1M30 exhibited anactivity of suppressing food intake, the possible use of saidpolypeptide as a pharmaceutical was indicated.

The result of amino acid alignment of human, mouse and rat nesfatin-1,and the sites of nesfatin-1N23, nesfatin-1M30 and nesfatin-1C29 areshown in B of FIG. 18. It was shown that the amino acid sequences of thesites of nesfatin-1M30 are highly conserved among species.

Working Example 21 Construction of an EIA System and the Detection ofthe nesfatin-1 Peptide in the Hypothalamus Tissue

From the result with an antibody (nesfatin-1 IgG) against nesfatin-1 andan antibody (PC1/3 and PC2) against prohormone convertase in the cellsof the rat hypothalamus in Working Example 11, it was demonstrated,nesfatin and prohormone convertase were expressed in the same cells andnesfatin-1 is likely to be produced therein. In order to furtherinvestigate this, a competitive EIA system that detects nesfatin ornesfatin-1 was constructed, and a fractionation pattern by the reversephase HPLC of a sample extracted from the tissue of the rat hypothalamusand a pattern of nesfatin-1 prepared by synthesis were compared.

The competitive EIA system was created using nesfatin-1 IgG prepared inWorking Example 10 and a biotin-labelled nesfatin-1 polypeptide.Nesfatin-1 IgG dissolved in PBS at 10 μg/ml was aliquoted into a 96-wellELISA plate (SUMITOMO BAKELITE Co., Ltd.: MS-8596F) at 50 μl/well, whichwas sealed with a plate seal and allowed to stand at 4° C. overnight inorder to immobilize the antibody. After washing each well of theantibody-immobilized plate with PBS, PBS containing 10% bovine serumalbumin (BSA) was dispensed at 250 μl/well and the plate was allowed tostand at room temperature for 2 hours. Then, after each well of theplate was washed three times with PBS, the antibody-immobilized platewas prepared.

In order to prepare a labelled nesfatin-1, a cysteine residue was addedto the C terminal of nesfatin-1 (nesfatin-1 Cys: SEQ ID NO: 62). Thoughthe method of preparation was similar to that of Working Example 16, PCRin order to obtain a nucleic acid encoding nesfatin-1 Cys was carriedout using the following primer set:

Forward primer: (SEQ ID NO: 63)5′-GGTTCCGCGGGTCTGGTTCCGCGTGGTTCTCCTATCGATGTGGACAA GACCAA-3′ Reverseprimer: (SEQ ID NO: 64) 5′-GGTTGCGGCCGCTTAACACCTCTTCAGCTCATCCAGTCTCG-3′

Nesfatin-1 Cys expressed and purified as in the method of WorkingExample 16 was dissolved in 0.1M phosphate buffer (pH 6.0) containing 50mM 2-mercaptoethanolamine and 1 mM EDTA and treated at 37° C. for 90minutes. Then trifluoroacetic acid (TFA) was added to 0.1%, which wasplaced on a Sep-Pak C18 column (Waters). After washing the column with10 ml of aqueous solution of 0.1% TFA and 10% acetonitrile, it waseluted with 3 ml of an aqueous solution of 0.1% TFA and 60%acetonitrile. After the eluate was lyophilized, it was dissolved in 0.1M phosphate buffer (pH 7.0) to 5 mg/ml, and 20 mg/ml of biotin (LongArm) maleimide (VECTRO Lab.) dissolved in 1/40 volume ofdimethylformamide (DMF) was added and reacted at room temperature for 3hours. To biotin (Long Arm) maleimide-reacted nesfatin-1 Cys, TFA wasadded to 0.1%, and was applied to HPLC equipped with a reverse phase C18column (Nacalai Tesque Inc.: COSMOSIL (trade mark) 5C18-AR-300 20.0 mmI.D.×150 mm). While monitoring absorbance at a wavelength of 210 nm, itwas washed with an aqueous solution of 0.1% TFA and then an aqueoussolution of 20% acetonitrile containing 0.1% TFA until the absorbance ofthe eluate cannot be observed. Subsequently, an aqueous solution ofacetonitrile at a 20-60% gradient was run to obtain a fraction havingthe highest peak of absorbance at 210 nm. The fraction obtained waslyophilized and then dissolved in PBS, which was used as the labellednesfatin-1.

The labelled nesfatin-1 was dissolved to 1 μg/ml in PBS containing 2%BSA. For the creation of a standard curve, the recombinant nesfatin-1prepared in Working Example 16 was diluted to a concentration of 6000ng/ml in PBS containing 2% BSA, which was then diluted in PBS containing2% BSA at a common ratio of 2 and used (standard samples: 6000, 3000,1500, 750.0, 375.0, 187.5, 93.8 ng/ml). Fifty μl each of the preparedlabelled nesfatin-1 and the standard samples were placed into amicrotest tube and mixed, and 50 μl each of them was dispensed in a wellof the antibody-immobilized plate. As an example of a biological sample,50 μl of the cerebrospinal fluid as it is collected or the samplediluted 2-fold in PBS containing 2% BSA was mixed with 50 μl of thelabelled nesfatin-1 solution in a microtest tube, and 50 μl thereof wasaliquoted in wells of the antibody-immobilized plate (test sample).After the aliquoted antibody-immobilize plate was allowed to stand atroom temperature for 1 hour, the reaction sample in the well wasdiscarded, and washed three times in PBS containing 0.2% Tween 20. Then50 μl of avidin-peroxidase (Sigma, A7419-2mL) diluted 1/1,000 with PBScontaining 2% BSA and 0.2% Tween 20 was dispensed in each well and wasallowed to stand at room temperature for 30 minutes. After the reaction,the solution in each well was removed, and after washing four times inPBS containing 0.2% Tween 20, it was washed twice in TBS (50 mMTris-HCl, 0.15 M NaCl, pH 8.0). To each well of the antibody-immobilizedplate after washing, 50 μl of a peroxidase substrate, TMB (PIERCE:1-Step (TM) Turbo TMB), was added and reacted at room temperature for 30minutes. Then to each well, 50 μl of 0.5 N sulfuric acid was added tostop the reaction, absorbance at a wavelength of 450 nm and that at awavelength of 620 nm were measured by an absorbance plate reader, andthe absorbance at a wavelength of 620 nm was subtracted from that at awavelength of 450 nm (450 (Δ620) nm) to obtain the measured value.

The analysis of nesfatin-1 expression at the hypothalamus was carriedout by fractionation using HPLC with the peptide extracted from thetissue as the sample. The hypothalamus was excised from the brains ofeight rats, homogenized in 4 ml of an aqueous solution of 0.1% TFA by aTeflon (trade mark) homogenizer, and centrifuged at 10,000 rpm for 10minutes to collect the supernatant. After the collected supernatant wasfiltered with a filter (Millipore) having a pore size of 0.45 μm, it wasrun through the Sep-Pak C18 column (Waters), and the column was washedin 5 ml of an aqueous solution of 0.1% TFA. Then, to the column 3 ml ofan aqueous solution of 60% acetonitrile was added for elution, and theeluate was dried in an evaporator. The dried product was dissolved in0.8 ml of an aqueous solution of 0.1% TFA, centrifuged at 10,000 rpm for10 minutes, and 500 μl of the supernatant was injected to a HPLCinstrument equipped with a C18 reverse phase chromatocolumn (NacalaiTesque Inc.: COSMOSIL % C18-AR-II, 4.6 mm I.D.×250 mm). After injectingthe sample, an aqueous solution of 0.1% TFA was run through the columnat a flow rate of 1 ml/min while monitoring the absorbance of the eluateat a wavelength of 224 nM. After washing, while keeping the flow rate,acetonitrile at a 0-60% concentration gradient (A1%/min) was run in thepresence of 0.1% TFA to collect the eluate as 1 ml fractions. Afterfreezing the fractions obtained at −80° C., they were lyophilized, andthe samples after drying were each dissolved in 200 μl of PBS containing2% BSA, and nesfatin and nesfatin/nesfatin-1 were measured by acompetitive EIA method. Similarly, from the samples obtained by drying600 μl of the cerebrospinal fluid collected from eight rats, peptidesamples were prepared, fractionated by HPLC, and the eluated fractionswere examined by the competitive EIA system. Also, as the control, 80 μgof the recombinant nesfatin-1 peptide prepared in Working Example 16 wasdissolved in 100 μl an aqueous solution of 0.1% TFA, which was injectedinto HPLC, and the fractions in which nesfatin-1 was eluated weredetected.

<Result>

A standard reaction curve obtained by measuring the standard samples inthe competitive EIA system is shown in FIG. 19 A-1. It was demonstratedthat with increased concentrations of the reacted nesfatin-1, thebinding of the labelled nesfatin-1 is competitively inhibited and theabsorbance at 450 (A620) decreases, indicating that nesfatin-1concentrations in samples can be determined in this system. Thesensitivity of this system corresponds to 4.6 ng/tube (93 ng/ml) ofnesfatin-1. The result of the concentration of nesfatin-1 (nesfatin) inthe cerebrospinal fluid determined by this competitive EIA system isshown in FIG. 19 A-2. The values obtained by measuring the cerebrospinalfluid as it is or after diluting ½ (converted by the dilution factorafter measurement) are almost the same and indicated that about 230ng/ml of nesfatin-1 (nesfatin) is present.

b-1 of FIG. 19B shows a result in which a peptide sample extracted fromthe rat hypothalamus was fractionated by HPLC and the presence ofnesfatin-1 in the fractions were determined, and b-2 of FIG. 19B shows aresult in which a similar study was carried out using a peptide sampleextracted from the rat cerebrospinal fluid. In both b-1 of FIG. 19B andb-2 of FIG. 19B, a reaction peak which is likely to be nesfatin-1 wasnoted in the fractions from No. 44 and 45. In a result when therecombinant nesfatin-1 was fractionated by HPLC under a similarcondition, nesfatin-1 was eluted at No. 44 fraction, the factor of whichpresence was indicated from HPLC fractions of the hypothalamus and thecerebrospinal fluid by the competitive EIA system is believed to benesfatin-1.

Working Example 22 Study on the Effect of the IntraperitonealAdministration of the Partial Peptide (nesfatin-1M16, nesfatin-1M14,nesfatin-1M10M) of nesfatin-1M30 on Food Intake Control in Mice

In Working Example 20, the study on the effect of suppressing foodintake of a partial peptide derived from nesfatin-1 led to the inventionof nesfatin-1M30. Furthermore, in order to examine in further detail thesites having an activity of suppressing food intake, a partial peptideof nesfatin-1M30 was prepared from the structure of nesfatin-1M30comprising a 30-amino acid length, and an experiment was carried out tomeasure the amount of food intake when it was intraperitoneallyadministered to mice.

For nesfatin-1M16 comprising a 16-amino acid length, nesfatin-1M14comprising a 14-amino acid length and nesfatin-1M10M comprising a10-amino acid length which are partial peptides of mouse nesfatin-1M30,the preparation of synthetic peptides having the following sequences wasreferred to Biologica Co., Ltd. and were purified by HPLC to a purity of95% or higher.

nesfatin-1M16: (SEQ ID NO: 71)N-ProAspThrGlyLeuTyrTyrAspGluTyrLeuLysGlnValIle Glu-C nesfatin-1M14:(SEQ ID NO: 72) N-ValLeuGluThrAspProHisPheArgGluLysLeuGlnLys-Cnesfatin-1M10M: (SEQ ID NO: 73) N-LysGlnValIleGluValLeuGluThrAsp-C

Each peptide was prepared in physiological saline so as to contain 10pmol per 100 μl, which was used as the sample for administration, and asthe control sample physiological saline (Vehicle) alone was used. Thephysiological saline as the control and the peptide samples preparedwere intraperitoneally administered at 100 μl per mouse (6 animals pergroup). Mice used were male ICR mice (Nippon SLC) and the housingcondition and the experimental condition were similar to those inWorking Example 18.

Each mouse that received administration was placed in an individualcage, and during 0-3 hours after the administration the weight decreasedof the pellet food was measured to determine the amount of food intake.For testing of significant difference, analysis of variance was used.

<Result>

FIG. 20 shows the amount of food intake during 0-3 hours afteradministration when a partial peptide of nesfatin-1M30, nesfatin-1M16(M16), nesfatin-1M10M (M10M) or nesfatin-M14 (M14), wasintraperitoneally administered into mice. In all of the group thatreceived nesfatin-1M16 (M16), nesfatin-1M10M (M10M) or nesfatin-M14(M14) relative to the control group (Cont.) that received physiologicalsaline alone, a significant effect of suppressing food intake was noted.

Working Example 23 Study on the Effect of Human nesfatin-1M30 and MouseNUCB1-M30 on Food Intake Control

Working Example 20 demonstrated that mouse nesfatin-1M30 has an effectof suppressing food intake. Based on this, human nesfatin-1M30 wasprepared and studied on its effect on the food intake behavior whenadministered on mice. Nucleobindin-1 (NUCB1) is a factor that forms afamily having a high homology with NEFA/nesfatin in the amino acidsequence of peptides and the nucleotide sequence of the genes. Thus,NUCB1-M30 which is a site corresponding to nesfatin-1M30 of NUCB1 wascreated and was administered to mice to examine the effect on foodintake.

The preparation of human nesfatin-1M30 (SEQ ID NO: 39) and mouseNUCB1-M30 by chemical synthesis was referred to Biologica Co., Ltd. andwas purified by HPLC to a purity of 95% or higher.

Human nesfatin-1M30: (SEQ ID NO: 39)N-ProAspThrGlyLeuTyrTyrAspGluTyrLeuLysGlnValIleAspValLeuGluThrAspLysHisPheArgGluLysLeuGlnLys-C Mouse NUCB1-M30: (SEQ IDNO: 103) N-ProAspThrGlyLeuTyrTyrHisArgTyrLeuGlnGluValIleAsnValLeuGluThrAspGlyHisPheArgGluLysLeuGlnAla-C

Each of the prepared human nesfatin-1M30 and mouse NUCB1-M30 wasprepared in physiological saline so as to contain 10 pmol per 100 μl,which was used as the sample for administration. For nesfatin-1M30 thatexhibited an activity of suppressing food intake, one prepared inWorking Example 20 was used as the comparative sample at similar amountsas nesfatin-1M30 and NUCB1-M30, and as the control sample physiologicalsaline (Vehicle) alone was used.

The physiological saline as the control and the peptide sample preparedwere intraperitoneally administered at 100 μl per mouse (n=6 per group).Mice used were male ICR mice (Nippon SLC) and the housing condition andthe experimental condition were similar to those in Working Example 18.

Each mouse that received administration was placed in an individualcage, and during 0-3 hours after the administration the weight decreasedof the pellet food was measured to determine the amount of food intake.For testing of significant difference, analysis of variance was used.

The alignment of amino acid sequence was performed for human, rat andmouse nesfatin and human, rat and mouse NUCB1. The method used the aminoacid sequences of human, mouse and rat nesfatin of SEQ ID NO: 2, SEQ IDNO: 5 and SEQ ID NO: 8, respectively, and the amino acid sequences ofhuman, mouse and rat NUCB1 of SEQ ID NO: 84, SEQ ID NO: 88 and SEQ IDNO: 92, respectively, and analyzed by the Clustal-W method.

<Result>

The result measured of the amount of food intake during 0-3 hours afteradministration when human nesfatin-1M30 (human/nesfatin-1M30), mousenesfatin-1M30 (mouse/nesfatin-1M30) or mouse NUCB1-M30 (mouse NUCB1) wasadministered is shown in FIG. 21A. FIG. 21A indicates that in all of thegroups that received human nesfatin-1M30 (human/nesfatin-1M30), mousenesfatin-1M30 (mouse/nesfatin-1M30) or mouse NUCB1-M30 (mouse NUCB1)relative to the control group (Vehicle) that received physiologicalsaline alone, a significant effect of suppressing food intake was noted.

Also, the results of alignment of amino acid sequence for human, rat andmouse nesfatin and for human, rat and mouse NUCB1 and sitescorresponding to nesfatin-1 and nesfatin-1M30 are shown in FIGS. 21B to21C. It was shown that amino acid sequences are highly conserved insites corresponding to nesfatin and nesfatin-1 of NUCB2, in particularthe site corresponding to nesfatin-1M30.

Working Example 24 Study on the Expression Site of the nesfatin Gene inthe Rat Hypothalamus

The expression of nesfatin mRNA in the brain hypothalamus was analyzedby an in situ hybridization method in Working Example 8. In order tofurther analyze the site where the nesfatin gene is being expressed, astudy by an in situ hybridization method using radioisotope was carriedout.

Using eight week-old male Wistar rats (purchased from Nippon SLC) (bodyweight: 220-250 g) that were housed with free access to the food, therats were deeply anesthetized by pentobarbital in the light period, andthe brain was fixed by perfusing 4% paraformaldehyde dissolved inice-cold 0.1M borate buffer (pH 9.5) from the heart. The brain wasextracted, and was immersed in 0.1M borate buffer (pH 9.5) containing10% sucrose and 4% paraformaldehyde for 2 days. The fixed brain wasfrozen in dry ice-acetone, and sliced with a cryostat to sections 20 μmthick, which were placed on a slide glass (MAS coat slide S-9116prepared by Matsunami Glass).

For the preparation of the radioisotope-labelled probe, a plasmid wasused which was obtained by cleaving a plasmid for the preparation of theNEFA probe that was used in Working Example 2 with a restriction enzymeNcoI and then purifying it. To 0.1 μg of the plasmid, 20 U (1 μl) of SP6RNA polymerase (Promega, P1085) was added at a condition of 19 μl of asolution containing 36 mM Tris-HCl buffer (pH 7.5), 6 mM magnesiumchloride, 2 mM spermidine, 8 mM dithiothreitol, 25 mM adenosinetriphosphate/guanosine triphosphate/cytosine triphosphate, 5 mM uraciltriphosphate and 5 mM [α-35S]-uracil triphosphate, and 1U of RNAsin™ribonuclease inhibitor (Promega, N2111), reacted at 37° C. for 60minutes to prepare a 35S-labelled NEFA cRNA probe. After the reaction,20 μl of the TNE buffer (10 mM Tris-C1, pH 8.0, 0.5 M NaCl, and 0.25 mMEDTA, pH 8.0) was added to stop the reaction, and then the probe waspurified using the NENSORB™ PREP Nucleic Acid Purification Cartridges(Perkin-Elmer, Inc., NLP028001EA) according to the attached protocol.

The slide glass of the prepared section sample was dried overnight undervacuum before hybridization, treated with PBS containing 10 μg/ml ofProtease K (Sigma, P2308) at 37° C. for 30 minutes, and washed twicewith PBS, and furthermore treated by immersing in 0.1 Mtriethylamine-hydrochloric acid buffer (pH 8.0) containing 0.25% aceticanhydride at room temperature for 10 minutes, followed by washing twicewith 2× concentrated SSC. The washed section sample was dehydrated byimmersing in 75% ethanol, 95% ethanol and 99% ethanol in this order,dried in the air, and further dried under vacuum.

On to the dried slide glass of the section sample, an in situhybridization solution (10 mM Tris-HCl buffer, pH 8.0, 30 mM NaCl, 10%dextran sulfate, 1× concentrated Denhardt's solution, 12 mM EDTA, 50%deionized formamide, 0.5 mg/ml yeast rRNA) was placed so as to cover thebrain tissue section, and prehybridized at 65° C. for 1 hour. Afterdiscarding the prehybridization solution, 80 μl, per slide glass, of ahybridization solution containing 10⁶ cpm/ml 35S-labelled NEFA cRNAprobe and 10 mM dithiothreitol was placed, and a cover slip was placedthereon, which was placed in a wet chamber and hybridized overnight at65° C. After the slide glass after hybridization was washed four timeswith 4× concentrated SSC, it was treated with the TNE buffer (10 mMTris-HCl, pH 8.0, 0.5 M NaCl, and 0.25 mM EDTA, pH 8.0) containing 20μg/ml of RNAase A at 37° C. for 30 minutes, washed twice with 2×concentrated SSC at room temperature, and further washed twice with 0.1×concentrated SSC at 65° C. for 30 minutes. The slide glass after washingwas dehydrated by immersing in 75% ethanol, 95% ethanol and 99% ethanolin this order, and then dried in the air. After the slide glass havingthe tissue section thereon was exposed to X-ray film for 7 days, it wasimmersed in a 2× diluted light-sensitive emulsion (Kodak, NTB3) andexposed to light for 3 weeks. After exposure the slide glass was washedwith water, stained with Thionine, and black spots resulted from theexposure were examined under microscope.

The position of each part in the rat brain was identified according toThe Rat Brain in Stereotaxic Coordinates by Paxinos G. and Watoson C.(Academic Press) (USA) 1986.

<Result>

A of FIG. 22 shows an image of in situ hybridization of a tissue sectioncontaining paraventricular nucleus (PVN) and supraoptic nucleus (SON), Bof FIG. 22 shows that of a tissue section containing zona incerta (Zi)and arcuate nucleus (Arc), and C of FIG. 22 shows that of a tissuesection containing lateral hypothalamic area (LHA). At each area of PVN,SON, Zi, Arc and LHA, spots that were light-sensitized by thehybridization of the radioisotope-labelled NEFA cRNA probe, indicatingthe expression of the NEFA gene.

Working Example 25 Study on the Effect of IntraventricularAdministration of nesfatin on Food Intake Behavior

While the amount of food intake during 0-1 hour/1-3 hours/3-6 hoursafter the administration of nesfatin to rats was studied in WorkingExample 6, the effect on food intake behavior during 6-12 hours afterthe administration was further studied herein.

In order to demonstrate the reproducibility of Working Example 6, theamount of food intake during 0-1 hour after administration wasdetermined when 0 (PBS alone), 1, 4 and 20 pmol of recombinant nesfatinwas administered to the third ventricle of brain, as in Working Example6. Also, in the same manner as in Working Example 6, after 5 pmol ofnesfatin was administered into the rat ventricle, the amount of foodintake was determined during 0-1 hour/1-3 hours/3-6 hours after theadministration. The control group used was the one that only receivedphysiological saline (0 pmol). For testing of significant difference,analysis of variance was used.

<Result>

As shown in A of FIG. 23, the amount of food intake at 0-1 hour afterthe administration of 4 pmol or 20 pmol of nesfatin into the brainventricle was significantly reduced as compared to the control group (0pmol) (p<0.01). Also, as shown in B of FIG. 23, a significant reductionin the amount of food intake was noted in the individual that receivedthe intraventricular administration of 5 pmol of nesfatin during 0-1hour (p<0.01), 1-3 hours (p<0.05) and 3-6 hours (p<0.001) as compared tothe control group (0 pmol). However, in the amount of food intake during6-12 hours, no difference was noted between the group that received 5pmol of nesfatin and the control group.

Working Example 26 Study on the Amount Expressed of nesfatin mRNA andthe nesfatin Polypeptide in the Rat Hypothalamus During Starvation

In order to study the expression of the nesfatin gene during starvation,in situ hybridization was carried out in the brain hypothalamus regionof the rats that had free access to the food and the fasted rats, andincreases or decreases in the amount of nesfatin mRNA at arcuatenucleus, paraventricular nucleus, lateral hypothalamic area andsupraoptic nucleus from the tissue were determined. Also, in order tostudy the amount expressed of the nesfatin-1 peptide at paraventricularnucleus, the hypothalamus region of the rats that had free access to thefood and the fasted rats was excised, and the extracted peptides weredetermined by competitive EIA system.

As in Working Example 8, rats used were individuals (the control group)that were allowed free access to the powder food, and individuals (thefasting group) that were housed on water alone without any food for 48hours. For the determination of the amount expressed of nesfatin mRNA ateach region of the hypothalamus, tissue sections were prepared in amanner similar to Working Example 24, which was subjected to in situhybridization, and the image obtained by exposing to X-ray film wasmeasured using an image analyzer (Imaging Research Inc., MCID™ Elite)(Imaki et al., Brain Research, Netherlands, 1993, Vol. 623, pp.223-228). Density of the exposed image at each region of arcuatenucleus, paraventricular nucleus, lateral hypothalamic area andsupraoptic nucleus was determined, and the values were changed intonumerical values as relative optical values on a 256-stage gray scalefrom white to black, which were then changed into numerical values asrelative optical densities according to the following equation to obtainthe absolute values for mRNA expression:

Relative optical density=log 10(256/relative optical value).

Each region of arcuate nucleus, paraventricular nucleus, lateralhypothalamic area and supraoptic nucleus in the rat brain was identifiedaccording to The Rat Brain in Stereotaxic Coordinates by Paxinos G. andWatoson C. (Academic Press) (USA) 1986.

The nesfatin-1 peptide at the paraventricular nucleus of the rat brainwas determined as follows. Rats that were allowed free access to thefood and that were fasted as described above were sacrificed bydecapitation, and the brain was immediately frozen in dry ice-ethanol,and sliced by a cryostat into sections 60 μm thick. The tissue was Nisslstained, and the parts corresponding to paraventricular nucleus on bothsides of the brain were excised and recovered. The recovered tissue washomogenized in 100 μl of 0.1 N hydrochloric acid in a 1.5 ml microtubeusing the microtube pestle (Scientific Specialties, 1005-39). Thehomogenized solution was centrifuged in a microfuge at 15,000 rpm for 20minutes, the supernatant was collected and the solvent was removed bylyophilization. The lyophilized sample was dissolved in 100 μl of PBS,and the amount of the nesfatin-1 peptide was determined at a conditionof 50 μl/well by the competitive EIA described in Working Example 21.

<Result>

A of FIG. 24 shows the result of image analysis by in situ hybridizationof nesfatin mRNA expression at various hypothalamus regions of arcuatenucleus (Arc), paraventricular nucleus (PVN), lateral hypothalamic area(LHA) and supraoptic nucleus (SON) in the rat brain in the feeding adlibitum group (control group) and the fasting group. In arcuate nucleus(Arc), lateral hypothalamic area (LHA) and supraoptic nucleus (SON), nodifference in the value (relative optical density) of nesfatin mRNAexpression was noted in the fasting group relative to that in thefeeding ad libitum group (control group). In contrast, in the result forparaventricular nucleus (PVN), the amount expressed of nesfatin mRNA inthe fasting group was significantly reduced relative to the feeding adlibitum group (control group).

B of FIG. 24 shows the result of image analysis by a competitive EIAmethod of the expression of the nesfatin-1 peptide at paraventricularnucleus (PVN) among the hypothalamic regions of the rat brain in thefeeding ad libitum group (control group) and the fasting group. It wasdemonstrated that the expression of the nesfatin-1 peptide atparaventricular nucleus is significantly decreased in the fasting grouprelative to the feeding ad libitum group (control group).

The above result shows that fasting markedly reduces the expression ofthe nesfatin (NEFA) gene and nesfatin-1 at paraventricular nucleus ofthe hypothalamus that is important in the control of food intake.

Working Example 27 Study on the Effect of IntraventricularAdministration of nesfatin-1 into the Rat Brain on Food Intake Behavior

It was shown in Working Example 12 that the part of nesfatin-1 alone hadan activity of suppressing food intake in the experiment of theadministration of partial peptides of nesfatin. In order to furthervalidate the experiment, changes in the activity of suppressing foodintake with time after administration was investigated.

The experimental condition was similar to that in Working Example 12,and 5 pmol of nesfatin-1 was administered into the third ventricle ofthe rat brain immediately before the start of the dark period, theamount decreased of the food (the amount of food intake) during 1 hourimmediately after administration (0-1 hr), during 2 hours from 1 hourafter the administration (1-3 hr), during 3 hours from 3 hours after theadministration (3-6 hr), and during 6 hours from 6 hours after theadministration (6-12 hr) was measured as the amount of food intake. Asthe control group, the amount of food intake by individuals thatreceived physiological saline alone was measured.

<Result>

FIG. 25 shows the amount of food intake during 0-1 hr, 1-3 hr, 3-6 hrand 6-12 hr when nesfatin-1 was administered to the third ventricle ofthe rat brain. Significant decreases in the amount of food intake in 0-1hr, 1-3 hr and 3-6 hr were noted in the nesfatin-1-administration grouprelative to the control group (p<0.01). In contrast, in 6-12 hr, anincrease in the amount of food intake was noted in thenesfatin-1-administration group relative to the control group (p<0.01).The above results demonstrated that the suppression of food intake bythe intraventricular administration of nesfatin-1 is temporary, and thedisappearance of the pharmacological effect of nesfatin with the passageof time and the resultant recovery of food intake can be noted.

Working Example 28 Study on the Specificity of Effect ofIntraventricular Administration of Anti-nesfatin-1 Antibody on EnhancingFood Intake

It was shown in Working Example 14 that the effect of enhancing foodintake is noted when an antibody against nesfatin-1 is administered intothe rat ventricle. In order to validate that the effect is due to theinhibition of the effect of nesfatin-1, a study was carried out todetermine whether the suppression of food intake by the intraventricularadministration of nesfatin-1 can be inhibited by the simultaneousadministration of anti-nesfatin-1 antibody.

The experimental condition was similar to that in Working Example 12,and for a group that received 5 pmol of nesfatin-1 alone into the thirdventricle of the rat brain immediately before the start of the darkperiod and a group that received 5 pmol of nesfatin-1 and 8 μg ofanti-nesfatin-1 antibody (nesfatin-1 IgG), the amount of food intake for1 hour after administration was measured and compared to that for thecontrol group (physiological saline alone was administered). Also, for agroup that received only leptin (Rat leptin: R & D Systems, 598-LP-01M)(1 μg) which is known to have an activity of suppressing food intake byintraventricular administration and a group that received leptin andanti-nesfatin-1 antibody, its effect on food intake was also studied.

<Result>

FIG. 26 is a graph showing the amount of food intake for 1 hour afteradministration for a group that received nesfatin-1 alone (nesfatin-1IgG/nesfatin-1/leptin: −/+/−), a group that received nesfatin-1 andanti-nesfatin-1 antibody (nesfatin-1 IgG/nesfatin-1/leptin: +/+/−), agroup that received leptin (nesfatin-1 IgG/nesfatin-1/leptin: −/−/+),and a group that received leptin and anti-nesfatin-1 antibody(nesfatin-1 IgG/nesfatin-1/leptin: +/−/+). The amount of food intakesignificantly decreased in the group that received nesfatin alonerelative to the control group (nesfatin-1 IgG/nesfatin-1/leptin: −/−/−),whereas in the group that received nesfatin-1 and anti-nesfatin-1antibody, the effect of enhancing the amount of food intake to about thesame degree as the control group was noted. In contrast, in the groupthat received leptin, the amount of food intake also decreased, whereasin the group that received leptin and anti-nesfatin-1 antibody, noeffect of enhancing the suppression of the amount of food intake byleptin was noted. This result suggests that the effect of enhancing foodintake by anti-nesfatin-1 antibody results from the specific suppressionof the effect of nesfatin-1.

Working Example 29 Study on the Binding Specificity of Anti-nesfatin-1Antibody

It was shown in Working Example 28 that the intraventricularadministration of anti-nesfatin-1 antibody specifically inhibits theeffect of nesfatin-1. Its binding property with other factors known atpresent to have an activity of controlling food intake was furtherstudied by the Western blotting method using extracts of the rat brain.

Western blotting analysis was carried out by the method described inWorking Example 3 with the primary antibody being changed from anti-NAPpolyclonal antibody to anti-nesfatin-1 antibody. Before reactinganti-nesfatin-1 antibody to the membrane in Western blotting as anexperiment for investigating the binding specificity of anti-nesfatin-1antibody, 5 μg of each peptide of NAP1-Ab (Working Example 10), andleptin (Rat leptin: R & D Systems, 598-LP-01M), αMSH (MelanocyteStimulating Hormone; Peptide Institute, Inc., 4057-v), CART (RatCocaine- and Amphetamine-Regulated Transcript 55-102; Peptide Institute,Inc., 4351-s), NPY (Human, Rat Neuropeptide Y; Peptide Institute, Inc.,4158-v), MCH (Human Melanin-Concentrating Hormone; Peptide Institute,Inc., 4369-v) and Orexin-A (Human, Rat, Mouse, Bovine Orexin-A; PeptideInstitute, Inc., 4346-s) per 1 μg of anti-nesfatin-1 antibody was added,and reacted at room temperature for 1 hour, and then Western blottingwas carried out in the method described above to examine whether or notthe bands disappear.

<Result>

A of FIG. 27 shows an image of Western blotting carried out withanti-nesfatin-1 antibody using protein extracts from the rat brain. As aresult, a band was noted at a position of molecular weight correspondingto 47.5 kd nesfatin polypeptide. B of FIG. 27 shows an image at about47.5 kd of Western blotting carried out after anti-nesfatin-1 antibodyand various peptides were previously reacted. When anti-nesfatin-1antibody was reacted to NAP1-Ab, the 47.5 kd band disappeared,indicating that the previous binding of NAP-1 Ab to NAP1-Ab peptideblocked the binding site of nesfatin. In contrast, when it was reactedto letptin, αMSH, CART, NPY, MCH and Orexin-A, and when the peptide wasnot subjected to the anti-nesfatin-1 antibody reaction, the 47.5 kb banddid not disappear. These results demonstrated that anti-nesfatin-1antibody specifically binds to nesfatin, but not to other foodintake-related peptides such as letptin, αMSH, CART, NPY, MCH andOrexin-A.

Working Example 30 Study on the Expression Site of nesfatin in the RatMedulla Oblongata

In Working Example 4, expression in the vicinity of the hypothalamus ofa rat brain was studied by immunohistochemical analysis. In order tofurther study the expression of nesfatin in the vicinity of the ratmedulla oblongata, a similar immunohistochemical analysis was carriedout.

Using eight week-old Wistar rats (purchased from Nippon SLC), braintissue sections of the part containing the medulla oblongata wereprepared from samples similarly to Working Example 4. The method ofimmunohistochemical stain was also similar to that in Working Example 4.Furthermore, the NAP peptide (Working Example 3) was added to anantibody (1 μg/ml) against the NAP peptide to 100 μg/ml, reacted at roomtemperature for 1 hour, and then immunohistochemical stain was alsocarried out using the antibody.

<Result>

A of FIG. 28 shows an image of immunohistochemical stain in the braintissue containing the medulla oblongata. In the immunohistochemicalstain in the brain tissue containing the medulla oblongata, stain wasnoted at STN: the nucleus of solitary tract, and the expression of thenesfatin polypeptide was noted. B of FIG. 28 shows an image ofimmunohistochemical stain carried out after an antibody against the NAPpeptide and the NAP peptide were previously reacted. Since the stainobserved in A of FIG. 28 disappeared by reacting the anti-NAP antibodyto the NAP peptide in advance, it was demonstrated, the nesfatinpolypeptide has been specifically stained in this immunohistochemicalstain. The results revealed that nesfatin is also expressed in thenucleus of solitary tract which is a viceral sensory nerve nucleus andis though to be involved in the mechanism of food intake control.

Working Example 31 Effect of Troglitazone Administration on Blood Leptinand the Intracerebral Expression of nesfatin in the Normal andLeptin-Resistant Obese Rats

In Working Example 2, the induction of the nesfatin (NEFA) gene incultured cells by troglitazone, a PPARγ agonist that was used as anantidiabetic drug, was studied. In order to further study the inducedexpression of nesfatin by troglitazone, troglitazone was given to ratsto study the induction of nesfatin in the brain. Similarly, blood levelsof leptin which is known to be responsible for control of food intakewere studied. In the study, normal Zucker rats (Zucker +/+: Lean) andZucker fa/fa, an animal model of leptin-resistant obese rats, were used.

As animals, 8 week-old male Zucker fa/fa (Zucker) rats and Zucker+/+(Lean) rats as the control animal were purchased from Nippon CharlesRiver, and were housed in a cycle of 12 hours of the light period from 6a.m. to 6p.m. and 12 hours of the dark period from 6 p.m. to 6 a.m. thenext morning and fed a powder food (Nippon Clea, CE-2), and kept at 22°C. After preliminary housing for over one week, individuals weighing200-250 g were selected among 9 to 10 week-old individuals from the ratspurchased and 6 animals per group were used in the experiment.Troglitazone (TGZ; Sankyo Co., Ltd.) was administered at a concentrationof 0.2% blended in the powder food, to which the animals were allowedfree access. As the control that did not receive troglitazone, rats thatwere housed on the normal powder food alone were used. After housing onthe troglitazone-containing food or the normal food for 10 days on end,the animals were weighed and sacrificed to collect whole blood. From therat's blood collected, serum was separated, and leptin concentration inthe serum was determined using a commercial ELISA kit (YanaiharaInstitute Inc., YK050) according to the attached protocol. Furthermore,the brain was harvested from the rats sacrificed, and was subjected toWestern blotting in a similar manner to Working Example 3, the densityof the bands was analyzed by an image analyzer (Imaging Research Inc.,MCID™ Basic), and the degree of color development of the band wasexpressed as relative units.

<Result>

A of FIG. 29 is a graph showing the body weight of the group (TGZ: +) inwhich the troglitazone-containing food was given to the normal rats(Lean) and the Zucker fa/fa rats (Zucker), and the group (TGZ: −) inwhich the troglitazone-free food was given thereto. In the normal ratsand the Zucker fa/fa rats as well, no marked difference in body weightdue to troglitazone administration was noted. Also, irrespective of theadministration of troglitazone, the Zucker fa/fa rats weighedsignificantly heavily (p<0.01) relative to the normal rats.

B of FIG. 29 is a graph showing the concentration of leptin in the bloodof the group (TGZ: −) in which the troglitazone-containing food wasgiven to the normal rats (Lean) and the Zucker fa/fa rats, and the group(TGZ: −) in which the troglitazone-free food was given thereto. In thenormal rats and the Zucker fa/fa rats, the concentration of leptin inthe blood significantly decreased (p<0.05) due to troglitazoneadministration. Also, irrespective of the administration oftroglitazone, the concentration of leptin in the blood was significantlyhigh in the Zucker fa/fa rats (p<0.01) relative to the normal rats.

C of FIG. 29 is a graph showing, as relative values, the density ofbands obtained by Western blotting of protein-extracted samples of thebrains from the group (TGZ: +) in which the troglitazone-containing foodwas given to the normal rats (Lean) and the Zucker fa/fa rats and thegroup (TGZ: −) in which the troglitazone-free food was given thereto. Inthe normal rats, no difference in the amount expressed of nesfatin inthe brain was noted between the group (TGZ: +) in which thetroglitazone-containing food was given and the group (TGZ: −) in whichthe troglitazone-free food was given. In contrast, in the Zucker fa/farats, the amount expressed of nesfatin in the brain significantlyincreased (p<0.01). Also, irrespective of the administration oftroglitazone, the amount expressed of nesfatin in the brain wassignificantly high in the Zucker fa/fa rats (p<0.01) relative to thenormal rats.

The above results suggest that troglitazone that was used as anantidiabetic drug does not induce nesfatin in the brain of the normalanimals, whereas it can induce the expression of nesfatin in the brainof the animal model that exhibits a leptin-resistant pathology. Incontrast, it is suggested, the enhanced blood concentrations of leptincannot be obtained by troglitazone in both the normal animals and thepathogenic animals, and conversely blood concentrations are decreased.

Working Example 32 Detection of the nesfatin-1 Peptide in the HPLCFractions of the Hypothalamus Tissue Extract

The peptide extracted from the hypothalamus tissue in Working Example 21as the sample was subjected to HPLC fractionation, and the fractionswere examined for the presence of nesfatin-1 by the competitive EIAassay system. Furthermore, in order to investigate the peptide detectedin the fractions is a molecule corresponding to nesfatin-1, the HPLCfractions of the hypothalamus tissue extract were analyzed by theWestern blotting method.

HPLC fractions of the hypothalamus tissue extract was prepared in asimilar manner to Working Example 21, and the fractions of No. (Fraction#) 43-47 were lyophilized. Samples after lyophilization were dissolvedin 100 μl of PBS, and 20 μl of it was subjected to SDS-PAGE on a 12%polyacrylamide gel, and then to Western blotting with anti-nesfatin-1antibody in a similar manner to Working Example 3.

<Result>

A of FIG. 30 shows an image of Western blotting of the fraction No. 45obtained by fractionating a peptide extract from the rat hypothalamus byHPLC. The molecular weight of the band detected by Western blotting isabout 9.7 kd, which almost agreed with that of the recombinantlyprepared nesfatin-1 peptide (Working Example 10, FIG. 9C). B of FIG. 30shows an image of the region at a molecular weight of about 9.7 kd inthe image of Western blotting of the fraction Nos. 43-47 obtained byfractionating the peptide extract from the rat hypothalamus by HPLC. The9.7 kd band is most strongly observed in fraction No. 45, which resultagreed with the pattern obtained by determining by a competitive EIAmethod the fractions obtained by fractionating the peptide extract fromthe rat hypothalamus by HPLC (b-1 of FIG. 19B).

The above result indicated that a molecule corresponding to nesfatin-1is present in the rat hypothalamus, and that a molecule corresponding tonesfatin-1 can be detected by fractionating it according to the methodin Working Example 21 and then by detecting by a competitive EIA methodand/or the Western blotting method.

Working Example 33 Study on the Specificity of Immunohistochemical Stainof the Regions of the Rat Hypothalamus

In Working Example 4, in order to analyze the NEFA expression site inthe hypothalamus of the rat brain which is related to food intake thecontrol, an immunohistochemical analysis using rat brain sections wascarried out. In order to study whether the stain is nesfatin-specific,an immunohistochemical analysis was carried out again taking intoconsideration the binding property of a peptide known to control foodintake to an antibody.

In a method similar to that in Working Example 4, immunohistochemicalstain was carried out on tissue sections of the rat brain eachcontaining arcuate nucleus and paraventricular nucleus, in whichanti-nesfatin-1 antibody (nesfatin-1 IgG; Working Example 10) was usedin stead of a anti-NAP polyclonal antibody as the primary antibody.Also, before reacting the primary antibody with the tissue sample, 5 μgof each peptide of anti-nesfatin-1 antibody (Working Example 10), andleptin (Rat leptin: R & D Systems, 598-LP-01M), αMSH (MelanocyteStimulating Hormone; Peptide Institute, Inc., 4057-v), CART (RatCocaine- and Amphetamine-Regulated Transcript 55-102; Peptide Institute,Inc., 4351-s) and NPY (Human, Rat Neuropeptide Y; Peptide Institute,Inc., 4158-v), known for the effect of controlling food intake, per 1 μgof anti-nesfatin-1 antibody was added, and reacted at room temperaturefor 1 hour, and then used in the immunohistochemical stain to validatethe reaction specificity of the antibody.

<Result>

A of FIG. 31 shows the result of immunohistochemical stain section usingan anti-nesfatin-1 antibody in the rat brain tissue containing thearcuate nucleus, and the result of immunohistochemical stain carried outafter the primary antibody and various peptides were previously reacted.In A of FIG. 31, since a-1 shows that the arcuate nucleus of the ratbrain is immunologically stained with the anti-nesfatin-1 antibody anda-2 shows that when the anti-nesfatin-1 antibody is previously reactedwith the nesfatin-1 peptide the stain disappears, it was demonstratedthat the anti-nesfatin-1 antibody detects nesfatin being expressed inthe arcuate nucleus. In contrast, when the anti-nesfatin-1 antibody waspreviously reacted with leptin (a-3 in A of FIG. 31), αMSH (a-4 in A ofFIG. 31), CART (a-5 in A of FIG. 31) and NPY (a-6 in A of FIG. 31), theimmunological stain in the arcuate nucleus does not disappear,indicating that these peptides do not bind to the anti-nesfatin-1antibody, and thus it was demonstrated that in the immunohistochemicalstain said antibody does not react to these peptides.

B of FIG. 31 shows the result of immunohistochemical stain using ananti-nesfatin-1 antibody in the rat brain tissue section containing theparaventricular nucleus, and the result of immunohistochemical staincarried out after the primary antibody and various peptides werepreviously reacted. In B of FIG. 31, since b-1 shows that the arcuatenucleus of the rat brain is immunologically stained with theanti-nesfatin-1 antibody and a-2 shows that when the anti-nesfatin-1antibody is previously reacted with the nesfatin-1 peptide the staindisappears, it was demonstrated that the anti-nesfatin-1 antibodydetects nesfatin being expressed in the paraventricular nucleus. Incontrast, when the anti-nesfatin-1 antibody was previously reacted withleptin (b-3 in B of FIG. 31), αMSH (b-4 in B of FIG. 31), CART (b-5 in Bof FIG. 31) and NPY (b-6 in B of FIG. 31), the immunological stain inthe arcuate nucleus does not disappear, indicating that these peptidesdo not bind to the anti-nesfatin-1 antibody, and thus it wasdemonstrated that in the immunohistochemical stain said antibody doesnot react to these peptides.

In the above result, the expression of nesfatin is specifically detectedin the immunohistochemical stain using the anti-nesfatin-1 antibody,indicating that in the rat brain nesfatin is being expressed in thearcuate nucleus and the paraventricular nucleus.

Working Example 34 Study on the Effect of Continuous Administration ofnesfatin-1 into the Rat Ventricle on the Weight of the Adipose Tissue

In order to study the effect of nesfatin-1 on the amount of the adiposetissue, nesfatin-1 was continuously administered into the rat ventricleto analyze changes in the weight of the adipose tissue etc.

Nesfatin-1 (5 pmol per day) or physiological saline alone (the controlgroup) was administered into the third ventricle of a rat using anosmotic pump for 10 consecutive days (5 and 4 rats were used per group).Intraventricular administration into rats by the osmotic pump wascarried out in a manner similar to that in Working Example 13.

After the administration of nesfatin-1 or physiological saline alone for10 days, each rat was sacrificed, dissected to excise all of theabdominal subcutaneous adipose tissue, the epididymal adipose tissue(these are white adipose tissue) and the retroperitoneal brown adiposetissue, and the weight of them was measured. Also, the gastrocnemialmuscles of the bilateral hind legs were harvested and the weight of themwas measured. From the weight of the tissues measured, their ratio tothe body weight of each individual was determined (tissue weight/bodyweight, mg/g). For testing of significant difference, analysis ofvariance was used.

<Result>

FIG. 32 is a graph of the result showing the ratio of tissue weight ofeach adipose tissue (A-E) and the gastrocnemial muscle (F) obtained fromthe rats that were given nesfatin-1 or physiological saline alone for 10days relative to the body weight. In the abdominal subcutaneous adiposetissue (A of FIG. 32), the epididymal adipose tissue (B of FIG. 32) andthe mesenteric adipose tissue (C of FIG. 32), significant decreases inthe ratio of tissue weight to body weight were noted in thenesfatin-1-administration group relative to the control group thatreceived physiological saline alone. In the retroperitoneal adiposetissue (D of FIG. 32), the ratio of tissue weight to body weight tendedto decrease but no significant difference was noted relative to thecontrol group. Furthermore, in the brown adipose tissue (E of FIG. 32)and the gastrocnemial muscle (F of FIG. 32), no significant differencein the total tissue weight to the body weight was noted between thecontrol group and the nesfatin-1-administration group.

The above result indicated that nesfatin-1 has an effect of decreasingthe ratio of tissue weight of the white adipose tissue to body weight,i.e. body fat percentage. It was also shown that it does not affect theratio of tissue weight of the brown adipose tissue and the muscle tissueto body weight.

Working Example 35 Study on the Effect of Intraperitoneal Administrationof nesfatin-1 into the Rat on Blood Biochemical Parameters

It was investigated whether the activities of suppressing food intake,suppressing body weight gain or reducing the amount of the adiposetissue are associated with changes in blood sugar (blood glucose values)and lipid-related parameters (cholesterol values, triglyceride values).

As experimental animals, 7 week-old male c57BL/6J mice were purchasedfrom Nippon Clea, and were housed in a cycle of 12 hours of the lightperiod from 6 a.m. to 6 p.m. and 0.12 hours of the dark period from 6p.m. to 6 a.m. the next morning and were allowed free access to a pelletfood (Nippon Clea, CE-2), and kept at 22° C.

Into the abdominal cavity of the c57BL/6J mice, 10 nmol of recombinantmouse nesfatin-1 prepared in Working Example 16 dissolved in 100 μl ofphysiological saline was administered, and as the control samplephysiological saline alone was used. Using a tuberculin syringe equippedwith a 26G needle, 100 μl each of the sample was administered once intothe abdominal cavity of each mouse (5 animals per group), and the timeof administration was immediately before the start (6 p.m.) of the darkperiod.

Each mouse that received administration was placed in an individualcage, and during 0-3 hours after the administration the weight decreasedof the pellet food was measured to determine the amount of food intake.Also, 3 hours after the administration each mouse was sacrificed bydecapitation to collect whole blood, and serum was collected therefrom.For the serum, the contents of glucose, total cholesterol andtriglyceride were measured using commercial reagents for determination(KYOWA MEDEX, Determiner L GLUII, Determiner L TCII, Determiner L TGII).

For testing of significant difference, analysis of variance was used.

<Result>

FIG. 33 is a graph of the assay result of the amount of food intake,glucose, total cholesterol and triglyceride in the blood when nesfatin-1or physiological saline alone was intraperitoneally given to the mice.

Nesfatin-1 administration significantly reduced the amount of foodintake as compared to the control group. In contrast, little differencewas noted between the nesfatin-1-administration group and the controlgroup in glucose content in the blood (glucose in the figure) whichrepresents a blood sugar level, or lipid-related parameters such astotal cholesterol content (cholesterol in the figure), triglyceridecontent (triglyceride in the figure).

The above result suggests that the effect of nesfatin-1 is to suppressfood intake, suppress body weight gain and reduce the amount of theadipose tissue without causing changes in blood sugar levels and bloodlevels of lipid-related parameters.

In accordance with the present invention, a factor involved in foodintake control and/or body weight control can be obtained by using aPPARγ agonist. Also by using nesfatin and/or nesfatin-1, diseasesassociated with metabolic and food intake disorders such as obesity oradiposis and nervous hyperphagia, and diseases associated with adiposissuch as type 2 diabetes mellitus, impaired glucose tolerance,hypertension, hyperlipidemia, hyperuricemia, fatty liver, cardiacdiseases, cerebral vascular diseases, sleep apnea syndrome, orthopedicdiseases such as osteoarthritis, menstrual disorders and malignanttumors can be prevented or treated. Furthermore, by using a substancesuch as antibody that suppresses the activity of nesfatin or nesfatin-1,anorexia in post-surgery and/or cancer patients, and diseases associatedwith nutritional and feeding disorders such as cibophobia can beprevented or treated.

1.-33. (canceled)
 34. A method of obtaining a factor related to foodintake control and/or body weight control, said method comprising thesteps of: acting a thiazolidine dione compound having a PPARγ agonistactivity on a mammalian cell; and obtaining genes of which theexpression is induced by said compound, and selecting and identifying agene encoding a protein having a signal peptide from said obtainedgenes.
 35. A polypeptide consisting of an amino acid sequence set forthin any of SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or 107-115.
 36. Apolypeptide having an activity of suppressing food intake and/orsuppressing body weight gain, said polypeptide comprising an amino acidsequence having a homology of at least 60% with an amino acid sequenceset forth in any of SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or 107-115;or an amino acid sequence in which some of the amino acids have beendeleted, inserted or substituted in the amino acid sequence set forth inany of SEQ ID NOs: 13-15, 39-41, 65-73, 101-103 or 107-115.
 37. Thepolypeptide according to claim 36 wherein said activity of suppressingbody weight gain is an activity of suppressing body fat gain.
 38. Anucleic acid molecule encoding a polypeptide set forth in claim
 35. 39.A nucleic acid molecule consisting of a nucleotide sequence set forth inany of SEQ ID NOs: 18-20, 44-46, 74-82, 104-106 or 116-124.
 40. Anucleic acid molecule that hybridizes to a nucleotide sequence set forthin any of SEQ ID NOs: 18-20, 44-46, 74-82, 104-106 or 116-124, or apartial sequence thereof under a stringent condition, and that encodes apolypeptide having an activity of suppressing food intake and/orsuppressing body weight gain.
 41. The nucleic acid molecule accordingclaim 38 wherein said activity of suppressing body weight gain is anactivity of suppressing body fat gain.
 42. A vector comprising thenucleic acid molecule according to claim
 38. 43. A transformantcomprising the nucleic acid molecule according to claim
 38. 44. Apharmaceutical composition for suppressing food intake and/orsuppressing body weight gain, said composition comprising, thepolypeptide according to claim 35 or a partial peptide thereof, thevector according to claim 42, or the transformant according to claim 43,as an active ingredient.
 45. The pharmaceutical composition according toclaim 44 wherein said activity of suppressing body weight gain is anactivity of suppressing body fat gain.
 46. An antibody that binds to thepolypeptide according to claim
 35. 47. A substance that suppresses theactivity or production of the polypeptide according to claim 35 or theexpression of a gene encoding said polypeptide.
 48. A pharmaceuticalcomposition comprising the substance according to claim 47 for enhancingappetite or enhancing body weight gain.
 49. A transgenic non-humanorganism into which the nucleic acid molecule according to claim 38 isintroduced.
 50. The transgenic non-human organism according to claim 49wherein said transgenic non-human organism is a transgenic non-humananimal that exhibits the state of suppressed food intake or the state ofsuppressed body weight gain.
 51. A transgenic non-human animal in whichthe antibody according to claim 46 or the suppressing substanceaccording to claim 47 is introduced and in which appetite or body weightgain is enhanced.
 52. A method of producing the polypeptide according toclaim
 35. 53. An assay method of predicting or diagnosing the state ofenhanced food intake or enhanced body weight gain comprising the stepof: detecting the amount contained of a nucleic acid molecule comprisinga nucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20,44-46, 74-82, 104-106 or 116-124, or of a polypeptide comprising anamino acid sequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15,39-41, 65-73, 101-103 or 107-115 in a biological sample from a mammal.54. An assay kit for use in the assay method according to claim 53, saidkit comprising: at least one of a PCR primer, a probe or a DNA chip fordetecting a nucleic acid molecule comprising a nucleotide sequence setforth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 or116-124; or an antibody recognizing a polypeptide comprising an aminoacid sequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15, 39-41,65-73, 101-103 or 107-115, a standard peptide, or a modified peptide forthe binding competitive reaction.
 55. A method of screening atherapeutic or preventive agent having an effect of suppressing foodintake and/or suppressing body weight gain, said method comprising thesteps of: contacting a test substance with a mammalian cell, anddetecting the increased expression of a gene in said cell comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 or 116-124, or the increased amount of a polypeptideintracellularly contained in said cell or extracellularly secretedcomprising an amino acid sequence set forth in any of in SEQ ID NOs: 3,6, 9, 13-15, 39-41, 65-73, 101-103 or 107-115.
 56. A method of screeninga therapeutic or preventive agent having an effect of suppressing foodintake and/or suppressing body weight gain, said method comprising thesteps of: administering a test substance to a mammal, and detecting theenhanced expression of a gene comprising a nucleotide sequence set forthin any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82, 104-106 or 116-124 orthe enhanced production of a polypeptide comprising an amino acidsequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15, 39-41, 65-73,101-103 or 107-115 in a biological sample from said test animal.
 57. Amethod of screening a therapeutic or preventive agent having an effectof suppressing food intake and/or suppressing body weight gain, saidmethod comprising the steps of: administering a test substance to thenon-human animal according to claim 49, or to a knock-out non-humananimal wherein the entire region or part thereof of a gene comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 or 116-124 has been deleted, and detecting thesuppression of food intake or the suppression of body weight gain insaid non-human animal.
 58. A therapeutic or preventive agent having aneffect of suppressing food intake and/or suppressing body weight gain,said agent being obtained by the method according to claim
 55. 59. Amethod of screening a therapeutic or preventive agent having an effectof enhancing food intake and/or enhancing body weight gain, said methodcomprising the steps of: contacting a test substance with a mammaliancell, and detecting the decreased expression of a gene comprising anucleotide sequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46,74-82, 104-106 or 116-124 in said cell, or the decreased amount of apolypeptide comprising an amino acid sequence set forth in any of SEQ IDNOs: 3, 6, 9, 13-15, 39-41, 65-73, 101-103 or 107-115 intracellularlycontained in said cell or extracellularly secreted.
 60. A method ofscreening a therapeutic or preventive agent having an effect ofenhancing food intake and/or enhancing body weight gain, said methodcomprising the steps of: administering a test substance to a mammal, anddetecting the suppressed expression of a gene comprising a nucleotidesequence set forth in any of SEQ ID NOs: 10-12, 18-20, 44-46, 74-82,104-106 or 116-124 or the suppressed production of a polypeptidecomprising an amino acid sequence set forth in any of SEQ ID NOs: 3, 6,9, 13-15, 39-41, 65-73, 101-103 or 107-115 in a biological sample fromsaid test animal.
 61. A method of screening a therapeutic or preventiveagent having an effect of enhancing food intake and/or enhancing bodyweight gain, said method comprising the steps of: administering a testsubstance to the non-human animal according to claim 49, or to aknock-out non-human animal wherein the entire region or part thereof ofa gene comprising a nucleotide sequence set forth in any of SEQ ID NOs:10-12, 18-20, 44-46, 74-82, 104-106 or 116-124 has been deleted, anddetecting the enhancement of food intake or the enhancement of bodyweight gain in said non-human animal.
 62. A therapeutic or preventiveagent having an effect of enhancing food intake and/or enhancing bodyweight gain, said agent being obtained by the method according to claim59.
 63. A pharmaceutical composition for suppressing food intake and/orsuppressing body weight gain, said composition comprising: thepolypeptide comprising an amino acid sequence set forth in any of SEQ IDNOs: 3, 6, 9, 13-15, 39-41, 65-73, 101-103 or 107-115, or a partialpeptide thereof; or the vector or transformant comprising the nucleicacid molecule comprising a nucleotide sequence set forth in any of SEQID NOs: 10-12, 18-20, 44-46, 65-73, 74-82, 104-106 or 116-124, as anactive ingredient.
 64. A pharmaceutical composition for enhancing foodintake and/or enhancing body weight gain, said composition comprising:an antibody that binds to the polypeptide comprising an amino acidsequence set forth in any of SEQ ID NOs: 3, 6, 9, 13-15, 39-41, 65-73,101-103 or 107-115; or a substance that suppresses the activity orproduction of the polypeptide or the expression of a gene encoding thepolypeptide.
 65. The polypeptide according to claim 35 wherein the aminoacid sequence is set forth in any of SEQ ID NOs: 13-15.
 66. Thepolypeptide according to claim 36 wherein the polypeptide consists of anamino acid sequence having a homology of at least 60% with an amino acidsequence set forth in any of SEQ ID NOs: 13-15, or of an amino acidsequence in which some of the amino acids have been deleted, inserted orsubstituted in the amino acid sequence set forth in any of SEQ ID NOs:13-15.
 67. A partial peptide of the polypeptide of claim 65, said thepartial peptide comprising an amino acid sequence set forth in any ofSEQ ID NOs: 39-41, 65-73, 101-103 or 107-115.
 68. The polypeptideaccording to claim 65 wherein at least one amino acid residue has beenchemically modified by a compound or a peptide.